CN101854951A - Formulations for TACI-immunoglobulin fusion proteins - Google Patents

Abstract

The invention relates to formulations of TACI-Immunoglobulin fusion proteins.

Description

The TACI-immunoglobulin fusion protein formulations

Invention field

The invention belongs to treatment protein formulation field.Say that more specifically the present invention relates to the pH scope is TACI-immunoglobulin (Ig) fusion protein formulations of 4.5-5.5.

Background of invention

BLyS ligand/receptor family

Identified already for BlyS (bone-marrow-derived lymphocyte stimulant) and two kinds of somatomedin of APRIL (a kind of part of inducing propagation) have three kinds of receptor: TACI (stride membrane activator and CAML-interact son), the BCMA (B cell maturation antigen) of unique combination affinity and BAFF-R (B cell activation factor receptor) (Marsters etc., 2000; .2001 such as Thompson).

TACI and BCMA can in conjunction with BLyS and APRIL the two, and BAFF-R it seems can only high-affinity in conjunction with BLyS (Marsters etc., 2000; .2001 such as Thompson).Therefore, BLyS can pass through all these three kinds of receptor conducted signals, and APRIL it seems can only be by TACI and BCMA conducted signal.In addition, the heterogeneous trimer compositions that has identified cyclicity BLyS and APRIL in general immunity rheumatisant's blood serum sample (is made up of three protein protomers, one or two copies containing BLyS and APRIL subunit), and prove that it can induce B cell proliferation (Roschke etc., 2002) external.

BLyS and APRIL are potent stimulant (Moore etc., 1999 of B cell maturation, propagation and survival; Schneider etc., 1999; Do etc., 2000).BLyS and APRIL are that autoimmune disease (autoimmune disease that particularly relates to the B cell) continues to exist necessary.Transgenic mice demonstration through engineered energy high level expression BLyS can be suffered from the immunocyte disease, and its symptom is similar to systemic lupus erythematosus patient finding (Gross etc., 2000; Mackay etc., 1999).Similarly, detect the rising of BLyS/APRIL level (Roschke, 2002 in systemic lupus erythematosus patient and other various autoimmune diseasees such as the rheumatoid arthritis patients serum sample; Cheema etc., 2001; Groom etc., 2002), thereby make BLyS and/or APRIL expand to the mankind from animal model with the related of B cell mediated diseases.The BLyS of patient's MS peripheral blood lymphocytes and T cell and APRIL up-regulated (Thangarajh etc., 2004; Thangarajh etc., 2005).Discovery is arranged near the spider cell BLyS that expresses the BAFF-R immunocyte and expresses rise (Krumbholz etc., 2005) strongly in the sick damage of MS.

Atacicept

Atacicept (INN) is a kind of recombination fusion protein, contains the outer ligand binding moiety of born of the same parents of receptor TACI (striding membrane activator and calcium regulator and cyclophilin-part (CAML)-interaction) and the human IgG Fc part of modification.The function of Atacicept is antagonism tumor necrosis factor (TNF) superfamily two member BLyS (B cell stimulatory agents) and APRIL (proliferation-inducing ligand).Proved that BLyS and APRIL are the important regulators of B cell maturation, performance function and survival.

Atacicept is a kind of human IgG that comprises ₁-Fc and a BLyS receptor TACI ectodomain part merge 313 amino acid whose soluble glycoprotein that form, and its estimated molecular weight is 35.4kDa.The conformation of this product is a dimer, and estimated molecular weight is 73.4kDa.Available recombinant technique produces Atacicept in Chinese hamster ovary (CHO) cell.

Human IgG among the Atacicept ₁-Fc is modified weakened that its Fc combines with the C1Q. component and with the interactional ability of antibody receptor (Tao etc., 1993; Canfield etc., 1991).These Fc effector functions that confirm Atacicept after testing weaken.

The treatment protein formulation

TACI-Ig fusion rotein such as atacicept are biological preparation, promptly treat the treatment albumen of human diseases, therefore can be to human administration.

Developing this preparation purpose provides treatment and proteicly effectively sends.Treatment with the problem that albumen usually runs into is, for example proteinic poor stability (often needing cold preservation or freezing preservation), and bioavailability difference and patient are unfriendly to the outer approach dosage form of intestines and stomach.

In the processing of biological technology products, need to obtain highly purified treatment protein solution usually.When preparing these protein solutions when (as sending), make protein stabilization most important for the parenteral road.Therefore, must select the excipient of energy stable protein.The stability of high-purity protein solution also may be subjected to the influence of buffer.Buffer influences the stability of protein in solution jointly by its ionic strength and pH.The buffer example that has been used for this purpose has phosphate, citrate, maleate and succinate buffer.

Even treatment albumen is solution when beginning to store, the problem that is run into is to keep that protein solution is stable to prevent that its cohesion when storing from forming granule or precipitation and preventing its degraded (as hydrolysis, oxidation, deacylated tRNA amine, by truncate or degeneration).

Temperature also can influence dissolubility, usually along with temperature rising dissolubility also raises.Yet protein can cause dissolubility to reduce or cohesion/precipitation by the part unfolding when being higher than certain temperature thresholding.

For preventing cohesion and degraded,, need exploitation to contain the stable therapeutic protein preparation of one or more excipient for obtaining medicine steady in a long-term.

The present invention is directed to this needs, the treatment albumen that can be used for treating human diseases is provided, promptly stable, pharmaceutically acceptable TACI-immunoglobulin fusion protein formulations.

Summary of the invention

The basis of foundation of the present invention is to have developed stable TACI-immunoglobulin fusion protein formulations.

First aspect, preparation of the present invention comprises:

A) TACI-immunoglobulin (TACI-Ig) fusion rotein, it comprises:

The ectodomain of i.TACI or its can be in conjunction with fragment or the variants of BLyS and/or APRIL; With

Ii. the constant region of immunoglobulin; With

B) make the pH scope of preparation maintain buffer between the 4.5-5.5.

Second aspect the present invention relates to comprise the pharmaceutical composition of this preparation.

The third aspect the present invention relates to be used for the treatment of or prevent the preparation of the present invention or the pharmaceutical composition of autoimmune disease or lymphocytic hyperplasia disease.

Fourth aspect the present invention relates to prepare the method for preparation of the present invention, and this method comprises the liquid solution of preparation TACI-Ig fusion rotein, and regulates the step of pH to the 4.5-5.5 scope of this liquid solution.

A fifth aspect of the present invention relates to the method for preparing preparation of the present invention, and this method is included in the step that fills the described preparation of scheduled volume in the sterilization container.

Detailed Description Of The Invention

The basis of foundation of the present invention is to have developed stable TACI-immunoglobulin fusion protein formulations, and the TACI-Ig fusion rotein in this preparation can be steady in a long-term (for example more than 3 months, more than 6 months, more than 12 months, more than 15 months or more than 18 months).

According to the present invention, described preparation comprises:

A) TACI-immunoglobulin (TACI-Ig) fusion rotein, it comprises:

The ectodomain of i.TACI or its can be in conjunction with fragment or the variants of BLyS and/or APRIL; With

Ii. the constant region of immunoglobulin;

B) make the pH scope of preparation maintain buffer between the 4.5-5.5.

In an embodiment of preparation of the present invention, the pH scope of described preparation is 4.7-5.3, more preferably 4.9-5.1.

Therefore the pH of described preparation can be 4.5,4.6,4.7,4.8,4.9,5.0,5.1,5.2,5.3,5.4 or 5.5.In a preferred embodiment, the pH of described preparation is 5.5.

The used buffer of preparation of the present invention can be, for example, and phosphate, acetate, citrate, succinate or histidine buffering liquid.The concentration range of described buffer can be 1-59mM, preferred 5-25mM.For example, the buffer concentration that comprises in the preparation of the present invention is 5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,25,30,35,40,45 or 50mM.

In a preferred implementation of preparation of the present invention, described buffer is an acetate buffer.Sodium acetate (NaAc) buffer preferably.In an embodiment of the invention, described buffer is 5-25mM, preferred 8-12mM, more preferably from about the NaAc buffer of 10mM.

In one embodiment, preparation of the present invention comprises excipient.Suitable excipient is, for example mannitol, sorbitol, glycerol or trehalose.The concentration range of mannitol or sorbitol can be (for example) 30-80 in the described preparation, or 40-60, or about 50 or 51mg/ml.The concentration range of glycerol can be (for example) 10-30 in the described preparation, or preferred 15-25, or 20 or 21mg/ml.

Trehalose is a kind of disaccharide, and by α, α-1,1 key connects to form by two glucose molecules.Trehalose in the preparation of the present invention, as anhydrous trehalose, its concentration range can be (for example) 50-120mg/ml, or preferred 60-100mg/ml.For example, preparation of the present invention can comprise 70,75,80,85,90,95,100,105 or the trehalose of 110mg/ml.In a preferred implementation, described preparation comprises the anhydrous trehalose of about 80mg/ml.

Though preparation of the present invention can comprise excipient or salt, as NaCl, CaCl ₂, MgCl ₂, but said preparation is preferred salt-free in description of contents of the present invention.

Preparation of the present invention also can comprise surfactant, as polysorbas20, or preferred Bo Luosamu (Poloxamer) 188 (

Or

F68).In one embodiment of the present invention, described preparation surfactant-free.

In one embodiment, preparation of the present invention also comprises antiseptic, and the combination of preferably adopting benzyl alcohol and benzalkonium chloride (benzalkonium chloride) is as antiseptic.For example, described preparation can contain the benzyl alcohol of 0.15-0.5%, benzyl alcohol as 0.2%, 0.3% or 0.4% and the benzalkonium chloride of 0.0007%-0.0015%, the benzalkonium chloride as 0.0008%, 0.0009%, 0.001%, 0.0011% or 0.0012%.In a highly preferred embodiment, described preparation contains the benzalkonium chloride of 0.3% benzyl alcohol associating 0.001%.

Preparation of the present invention comprises TACI-immunoglobulin (TACI-Ig) fusion rotein as therapeutical active compound, promptly as active component.Described TACI-Ig fusion rotein comprises the ectodomain of (a) TACI or it can be in conjunction with variant or the fragment of BLyS and/or APRIL; (b) constant region of immunoglobulin.Those skilled in the art will know that the TACI-Ig fusion rotein of making according to the present invention is not anti--TACI antibody.Any anti--TACI antibody can not comprise the ectodomain of TACI or its can be in conjunction with variant or the fragment of BLyS and/or APRIL, but at the epi-position in the TACI ectodomain.

In framework of the present invention, term " TACI ectodomain " also refers to have at least 80% or 85% with TACI ectodomain (SEQ ID NO:1), any variant of preferred at least 90% or 95% or 99% homogeny.Term " TACI ectodomain " also comprises the variant that contains no more than 50 or 40 or 30 or 20 or 10 or 5 or 3 or 2 or 1 conservative amino acid substitutions.Any this variant can be in conjunction with BlyS and/or APRIL, and/or the heterogeneous trimer of any BlyS-APRIL.Preferred this class variant also can suppress BlyS and/or APRIL and/or the heterogeneous trimerical biologic activity of any BlyS/APRIL.The biologic activity of described BlyS or APRIL is, for example, and B cell proliferation.

Among the present invention, also can adopt the fragment (active fragment) and the variant of TACI ectodomain, as long as these fragments can be in conjunction with BlyS and/or APRIL, and/or the heterogeneous trimer of BlyS-APRIL.Preferred this class fragment also can suppress or weaken the heterogeneous trimerical biologic activity of BlyS and/or APRIL and/or BlyS/APRIL.

Can be described by for example following examples 2, assess any TACI ectodomain, TACI-Ig fusion rotein or its any variant or fragment in conjunction with BlyS and/or APRIL and/or the heterogeneous trimerical ability of BlyS/APRIL.Can be described by for example following examples 3, assess the ability that they suppress or weaken BlyS, APRIL and/or the heterogeneous trimer biologic activity of BlyS/APRIL.

In the present invention, any this fragment or the variant of TACI ectodomain, or TACI-Ig fusion rotein preferably do not have and are starkly lower than the active any biologic activity of atacicept (protein that promptly has SEQ ID NO:3 aminoacid sequence).

Term used herein " immunoglobulin (Ig)-constant region " is also referred to as " Fc district ", and it is derived from human or animal's immunoglobulin (Ig), preferred IgG.IgG can be IGg1, IgG2, IgG3 or IgG4.The Fc district preferably includes CH2 at least, the CH3 district of IgG1, is preferably also to comprise hinge region.

The constant region of preferred Ig is the human IgG1 district.

In one embodiment, the modified cytotoxicity (CDC) of its complement dependence and/or the cytotoxicity (ADCC) that antibody relies on of having reduced of human IgG1's constant region.

In ADCC, the Fc district of antibody is incorporated into the Fc receptor (Fc γ R) on immune effector cell such as natural killer cell and macrophage surface, causes target cell to be engulfed or dissolve.In CDC, antibody kills target cell by trigger the complement cascade reaction at cell surface.According to the residue that is positioned on hinge region and the CH2 domain, IgG can and suppress receptor (Fc γ RIIb) Fc γ R in conjunction with activated receptor (Fc γ RI, Fc γ RIIa, Fc γ RIIIa and Fc γ RIIIb), or complement the 1st component (C1q).In IgG2 and IgG4, two zones in the CH2 domain in conjunction with most important, and have unique sequences to Fc γ R and C1Q..For example, the residue of IgG2 position 233-236 is substituted by human IgG1 (residue) and greatly reduces its ADCC and CDC activity (Armour etc., 1999 and Shields etc., 2001).According to EU index position (Kabat etc., 1991), can in the Fc district of IgG1, introduce following Fc sudden change:

T250Q/M428L

M252Y/S254T/T256E+H433K/N434F

E233P/L234V/L235A/ΔG236+A327G/A330S/P331S

E333A；K322A

Other Fc sudden change can be, for example in the replacement that is selected from 330,331,234 or 235 or its bonded EU index position.Among the present invention, also can in the Fc district, introduce the aminoacid replacement that is positioned at the EU index position 297 on the CH2 domain, connect the potential site that sugar adheres to eliminate N-.Also available serine residue replaces the cysteine residues of EU index position 220, to remove this cysteine residues common and immunoglobulin light chain constant district formation disulfide bond.

Prepared the specific Fc domain that is suitable for TACI-Ig fusion rotein of the present invention.

Specifically, for producing the Fc fusion rotein, prepared the human IgG1 Fc of 6 kinds of modifications, called after Fc-488 and Fc4, Fc5, Fc6, Fc7 and Fc8.

Utilize wild type human normal immunoglobulin γ 1 constant region to make template, designed and made up and be convenient to clone the Fc-488 (having the DNA sequence of SEQ ID NO:4 and the aminoacid sequence of SEQ ID NO:5) that contains people γ 1Fc district fusion rotein.Should note owing to replace the cysteine that forms disulfide bond usually with the immunoglobulin light chain constant district, may cause the unpaired and generation illeffects of cysteine residues with serine.In the codon of coding EU index position 218, introduce a kind of other and change, help the DNA operation of back to introduce BgIII Restriction Enzyme recognition site.By the PCR primer these are changed in the PCR product of introducing coding.Owing to settled the BgIII site, in order to finish (introducings) Fc hinge region, the codon of encode EU index position 216 and 217 is mixed in companion's sequence of fusion rotein.Fc4, Fc5 and Fc6 comprise sudden change, by weakening Fc γ R combination and complement C1g in conjunction with the effector functions that weakens the Fc mediation.Fc4 contains same aminoacid replacement, and they are introduced among the Fc-488.Introduce other aminoacid replacement, to weaken the effector functions of potential Fc mediation.Specifically, introduce three aminoacid replacement to weaken Fc γ RI combination, these replacements are the replacements in EU index position 234,235 and 237.The replacement that has shown these positions has weakened combine (Duncan etc., 1988) with Fc γ RI.These aminoacid replacement may also weaken with Fc γ R11a combine and with (Sondermann etc., 2000 of combining of Fc γ RIII; Wines etc., 2000).

Several research groups reported EU index position 330 and 331 aminoacid and C1Q. in conjunction with and fixing relevant (Canfield and Morrison, 1991 of follow-up complement; Tao etc., 1993).Introduce aminoacid replacement in these positions of Fc4 and weakened its complement fixation function.The CH3 district of Fc4 is identical with the CH3 district of corresponding wild type peptide, except termination codon.When cultivating clone's DNA, this termination codon is changed over TAA to eliminate potential harmful site that methylates from TGA with bad coli strain.

In Fc5, the arginine residues of EU index position 218 lysine that suddenlys change back is not because adopt BgIII clone scheme in containing the fusion rotein of this specific Fc.The remainder of Fc5 sequence and Fc4 described above are complementary.

Fc6 is identical with Fc5, except carboxyl terminal lysine codon has been eliminated.The lysine of maturation immunity globulin C-end is removed to come out from B emiocytosis then from translation after ripening immunoglobulin usually, or removes circulation time at serum.As a result, circulating antibody does not have the terminal lysine residue of C-usually.Identical with above Fc4 and Fc5, the termination codon in the Fc6 sequence changes over TAA.

Fc7 is identical with wild type γ 1Fc, has the aminoacid replacement on the EU index position 297 in the CH2 domain.EU index position 297 agedoites are sugared attachment points that N-connects.Because sugared structure may produce differences between batches, N-connects sugar and introduced potential variation source in recombinant expressed albumen.In the trial of a this potential variation of elimination, making 297 asparagine mutations is that glutamine residue is adhered to N-and connected sugar to prevent this residue position.The sugar at residue 297 places also participates in combine (Sondermann etc., the Nature 406:267 (2000)) of Fc and FcIII.Therefore, removing this sugar usually should weaken and contain combining of Fc7 recombination fusion protein and Fc γ R.As mentioned above, the termination codon in the Fc7 sequence is sported TAA.

Fc8 is identical with wild type immunoglobulin γ 1 district shown in the SEQ ID NO:4, except the cysteine of EU index position 220 is replaced by serine residue.Cysteine residues common and immunoglobulin light chain constant district formation disulfide bond has been eliminated in this sudden change.

The specific Fc of any of these district all falls into the scope of the invention in the application that forms the TACI-Ig fusion rotein.

The constant region for immunoglobulin of TACI-Ig preferably comprises the polypeptide with aminoacid sequence shown in the SEQ ID NO:2, or the Ig constant region has 80% or 85% at least shown in itself and the SEQ ID NO:2, preferred at least 90% or 95% or 99% identical variant; Or contain and be less than the variant that 50 or 40 or 30 or 20 or 10 or 5 or 3 or 2 conservative amino acid replace, as long as this does not influence the whole biologic activity of TACI-Ig fusion rotein, the proteic immunogenicity of this kind TACI-Ig can not be significantly higher than atacicept (SEQ ID NO:3).

In description of the present invention, term " homogeny " expression is by the relation between sequence more determined two or many peptide sequences.Homogeny is often referred to two peptide sequences and makes full length sequence relatively the time, and their aminoacid is accurate corresponding respectively with aminoacid.

For not having accurately corresponding sequence, can determine that it is " 0% is identical ".Usually arrange two sequences that contrast is compared, obtain the maximum correlation between the sequence.This can be included in one or two sequences and insert " space " to improve the contrast degree.The total length of more every sequence (what is called totally contrasts) can be determined homogeny %, and this is particularly suitable for the identical or closely similar sequence of length, or the sequence (so-called local contrast) shorter, that length limits, and this is fit to the unequal sequence of length.

Relatively the method for two or many sequence homogenies is well known in the art.For example, can adopt this program (Devereux J etc., 1984) in 9.1 editions Wisconsin sequence analysis bags, for example BESTFIT and GAP program, measure two between the polynucleotide homogeny % and the homogeny % between two peptide sequences.BESTFIT adopts " local homology " algorithm of Smith and Waterman (1981), seeks the best single area of similarity between two sequences.Other program of measuring the sequence homogeny also is known in the art, for example, and BLAST family program (Altschul S F etc., 1990; Altschul S F etc., 1997, can pass through the NCBI homepage Www.ncbi.nlm.nih.govEnter) and FASTA (Pearson W R, 1990).

It is the replacement that is known as " conservative " that preferred amino acids of the present invention replaces.The conservative amino acid of the TACI ectodomain of TACI-Ig fusion rotein or constant region for immunoglobulin part replaces, comprise the interior on the same group synonym aminoacid (replacing mutually) that physicochemical property is fully similar, replacement between the interior on the same group member will be preserved the biological function (Grantham, 1974) of this molecule.Known also can the manufacturing in above-mentioned sequence inserted and disappearance and its function that can not change, specifically, if insert or lack and only relate to a few amino acids, as below 50 or below 30, below 20 or preferred below 10 or 5 following amino acid residues, and do not remove or replace the aminoacid most crucial, as cysteine residues to functional conformation.Such disappearance and/or the protein that insertion produced and variant can be used for treating recurrent MS, as long as its biologic activity significantly is not lower than the biologic activity of atacicept (albumen with aminoacid sequence shown in the SEQ ID NO:3).

International Patent Application WO of having announced 00/40716 and WO 02/094852 disclose ectodomain and TACI ectodomain and its part BlyS and the interactional specific fragment of APRIL of TACI.

As WO 00/40716 was disclosed, the ectodomain of TACI comprised 2 repetitive sequences that are rich in cysteine (Cys), and this is the feature of tumor necrosis factor (TNF) receptor superfamily member under the taci receptor.Also prepared among the WO 00/40716 and a kind ofly can prune variant with the bonded TACI of BlyS, called after BR42x2, it only comprises the 2nd kind of more conservative Cys repetitive sequence that is rich in.Therefore, in framework of the present invention, the ectodomain fragment of TACI preferably comprises at least corresponding to the 71-104 amino acid residue of the 2nd kind of SEQ ID NO:1 that is rich in the Cys repetitive sequence or by these residues and forms, and more preferably the TACI-Ig fusion rotein also comprises the 34-66 amino acid residue that is rich in the SEQ ID NO:1 of Cys repetitive sequence corresponding to first kind.

In another embodiment of the present invention, described energy is in conjunction with self comprising the 30-110 amino acid residue of SEQ ID NO:1 with the ectodomain fragment that suppresses BlyS and/or the active TACI of APRIL or being made up of these residues.

Also have in the embodiment of the present invention, described TACI-Ig fusion rotein comprises SEQ IDNO:3 polypeptide of sequence or is made up of it, or at least 90% or 95% or 98% or 99% homogeny is arranged with it or be less than 30 or 20 or 15 or 10 or 5 or 3 or 2 variants that conservative amino acid replaces, this variant can be in conjunction with BlyS and/or APRIL.

Also have in another embodiment of the present invention, described TACI-Ig fusion rotein comprises SEQ IDNO:8 polypeptide of sequence or is made up of it, or at least 90% or 95% or 98% or 99% homogeny is arranged with it or be less than 30 or 20 or 15 or 10 or 5 or 3 or 2 variants that conservative amino acid replaces, this variant can be in conjunction with BlyS and/or APRIL.

Also have in the embodiment of the present invention, described TACI-Ig fusion rotein comprises SEQ IDNO:10 polypeptide of sequence or is made up of it, or at least 90% or 95% or 98% or 99% homogeny is arranged with it or be less than 30 or 20 or 15 or 10 or 5 or 3 or 2 variants that conservative amino acid replaces, this variant can be in conjunction with BlyS and/or APRIL.

Also have in the embodiment of the present invention, described TACI-Ig fusion rotein comprises SEQ IDNO:12 polypeptide of sequence or is made up of it, or at least 90% or 95% or 98% or 99% homogeny is arranged with it or be less than 30 or 20 or 15 or 10 or 5 or 3 or 2 variants that conservative amino acid replaces, this variant can be in conjunction with BlyS and/or APRIL.

Also have in the embodiment of the present invention, described TACI-Ig fusion rotein comprises SEQ IDNO:14 polypeptide of sequence or is made up of it, or at least 90% or 95% or 98% or 99% homogeny is arranged with it or be less than 30 or 20 or 15 or 10 or 5 or 3 or 2 variants that conservative amino acid replaces, this variant can be in conjunction with BlyS and/or APRIL.

In yet another embodiment of the present invention, described preparation comprises concentration range 20mg/ml-180mg/ml, and for example concentration is 25,30,35,40,45,50,55,60,65,70,75,80,85,90,95,100,105,110,115,120,125,130,135,140,145,150,155,160,165,170, the TACI-Ig fusion rotein of 175mg/ml.

In another embodiment of the present invention, described preparation is liquid (as an aqueous solution) dosage form.

Also have in the embodiment of the present invention, described preparation is the multiple dose administration preparation.Preferably comprise antiseptic in the multiple dose preparation.As mentioned above, one preferred embodiment in, described preparation comprises benzyl alcohol (as 0.3%) and benzalkonium chloride (as 0.001%).

But every day, every other day, preferably secondary or give the TACI-Ig fusion protein formulations once in a week weekly.Give TACI-Ig and preferably inject administration, weekly.Perhaps, also can week about or once give described preparation in every month.

Preparation of the present invention can be for example intravenous, subcutaneous or intramuscular administration.In an embodiment of the invention, described preparation is used for subcutaneous administration.

Preparation of the present invention is used for the treatment of disease, the preferred therapeutic human diseases.Therefore, in one embodiment, formulation preparation of the present invention is become pharmaceutical composition.

The preparation or the pharmaceutical composition that comprise the TACI-Ig fusion rotein are preferred for treating autoimmune disease or lymphocytic hyperplasia disease, or the pharmaceutical drugs of preparation treatment autoimmune disease or lymphocytic hyperplasia disease.

Autoimmune disease of the present invention includes but not limited to: systemic lupus erythematosus (SLE), lupus nephritis, rheumatoid arthritis, multiple scleroderma or optic neuritis.

The lymphocytic hyperplasia disease is a kind of disease of lymphsystem cell hyperproliferation.The B malignant change of cell is a lymphocytic hyperplasia disease for example.B malignant change of cell disease includes but not limited to: leukemia and lymphoma, for example, acute leukemia, acute lymphoblastic leukemia, acute myeloid leukemia, pith mother cells leukemia, promyelocytic leukemia, grain-monocytic leukemia, mononuclear cell erythroleukemia, chronic leukemia, chronic myelocytic (granulocyte) leukemia, chronic lymphocytic leukemia, polycythemia vera, Hodgkin, non Hodgkin lymphoma, multiple myeloma and macroglobulinemia Waldenstron.

The present invention also relates to produce or prepare the method for preparation of the present invention, comprise the step of preparation (for example mixing) component (a)-(c), preferably in liquid solution (as aqueous solution).

The invention still further relates to the method for producing or preparing preparation of the present invention, be included in the described preparation of placing scheduled volume in the sterilization container.Scheduled volume can be 0.5-5ml for example, preferred 1-2ml.

In an embodiment of the invention, described container is selected from vial or prefill syringe.For example available no coating bottle stopper of vial or the sealing of coating bottle stopper.Bottle stopper can be for example rubber closure or brombutyl rubber stopper.Syringe can clog with the rubber inner core or with the coating inner core as the prefill syringe.Coating can be for example not have oily silicone coating.

The prefill syringe can have different capabilities, as 0.5,1,1.5 or 2ml.Preferred 1ml syringe.Loading capacity of syringe preferred 1 or 1.2ml.The prefill syringe can be made of plastic, or be preferably the glass syringe.Suitable glass syringe is the 1mL glass syringe 27G1/2 RNG W 7974/50G that makes of Becton Dickinson for example.The prefill syringe preferably uses coating stopper (as the W4023/50G of FluroTec company manufacturing) and no coating stopper (as the W4023/50G of West Pharmaceutical company manufacturing) to clog.According to the present invention, the prefill syringe preferably comprises TACI-Ig fusion rotein content range at 20-160mg, and for example 20,25,50,75,100,125 or the medicine of 150mg.Shown in following examples 4, the TACI-Ig fusion protein formulations that comprises the pH5.0 of sodium acetate buffer and trehalose remains on 5-25 ℃ can be steady in a long-term, for example reaches 18 months.

Except as otherwise noted, the numerical value of the various scopes that present specification is specified all is expressed as approximation, as minimum in the described scope and peak front " approximately " this speech is arranged.By this way, can adopt on described scope or under slight change to reach the result substantially the same with the numerical value in the scope.Also have, disclosed scope is a successive range, comprises the minimum quoted and each value between the peak, and any scope that may form.

In the present invention, preparation of the present invention or pharmaceutical composition can comprise other active component except that the TACI-Ig fusion rotein, or with other active component administering drug combinations.For example, can there be corticosteroid, particularly medrat also has interferon-beta, carat Qu Bin, mitoxantrone, acetic acid glatiramer, natalizumab, Rituximab, teriflunomide, fingolimod, laquinimod or BG-12 (a kind of oral fumarate).This therapeutic alliance can be simultaneously, separately or carry out in succession.

Now disclose the present invention, those skilled in the art can understand, adopt far-ranging equal parameter, concentration and condition, do not deviate from thinking of the present invention and scope comprehensively, need not too much experiment and can obtain same result.

Though described the present invention in conjunction with specific embodiments, should understand and to be further improved.The application is intended in its claims scope to cover according to the principle of the invention any change, application and reorganization that the present invention did, comprises the known or conventional practice that belongs to field under the present invention, the content that openly departs from this description applicable to above-mentioned basic feature.

All lists of references that this paper quotes, comprise magazine article or summary, disclose or the undocumented U.S. or foreign patent application, the U.S. that issues or foreign patent and other any list of references, integral body is included this description in as a reference, comprises all data, chart and text in the incorporated by reference document.In addition, also to include this description in for referencial use for the list of references content in this description institute incorporated by reference document.

With reference to known method step, conventional method step, known method or conventional method, do not mean and admit that by any way any aspect of the present invention content, description or embodiment are disclosing, report or proposing to the each side field.

More than the description of the specific embodiment has been disclosed general characteristic of the present invention fully, other people can need not too much test and not deviate from general conception of the present invention by the knowledge (content that comprises list of references that this description is drawn) of using the present technique field, are not difficult to modify and/or adapt such specific embodiment for various application.Therefore, reorganization and the modification made of these explanations that provide according to this description and guide is intended to fall in the equal scope of disclosed embodiment.It is unrestricted for explanation also should understanding used word of this description and term purpose, therefore term of this description or the word explanation and the guide that can be provided according to this description by those skilled in the art makes an explanation in conjunction with those of ordinary skills' knowledge.

After having described the present invention, be understood that referring to the embodiment of following exemplary clinical research situation it is not in order to limit the present invention just for explanation provides.

The exploitation of embodiment 1. liquid preparations

Vocabulary

AUC: analytical type is super centrifugal,

CD: garden dichromatic

DLS: dynamic light scattering,

DSC: differential scanning calorimetry

IEC: ion exchange chromatography,

MALDI-ToF: substance assistant laser desorpted ionized time-of-flight mass spectrometry

OD: optical density,

RALS: right angle light scattering

RP: reversed phase chromatography,

SEC: molecular size exclusion chromatography

Material

-TACI-Fc medicine, phosphate buffer, pH6.0 ,+140mM NaCl

-TACI-Fc medicine, phosphate buffer, pH5

-TACI-Fc medicine, acetate buffer, pH5

-orthophosphoric acid (1.00563, Merck)

-succinic acid (1.00682, Merck)

-citric acid (1.59134, Merck)

-histidine (1.04351, Merck)

-glacial acetic acid 100% (1.00063, Merck)

-D-mannitol DMB, Ph Eur, BP, USP, FCC, E421 (1.05980, Merck)-the D-sorbitol (S-1876, Sigma)

-sucrose DAB, Ph Eur, BP, NF (1.07653, Merck)

-trehalose (1.08216, Merck)

-D-Dextrose monohydrate (346971, Carlo Erba)

-sodium hydroxide sheet GR (1.06498, Merck)

-polysorbas20, synthetic (8.22184, Merck);

-poloxamer (Poloxamer) 188 (Lutrol F 68 DAC, USP/NF, Basf)

-calcium chloride dihydrate (1.02382, Merck)

-magnesium chloride (1.05833, Merck)

-sodium chloride (1.06404, Merck)

-arginine hydrochloride (A-5131, Sigma);

-lysine hydrochloride (1.0571, Merck)

-glycine (5.00190, Merck);

-acetonitrile (00030, Merck)

-10xPBS(P/N?70013-032，Gibco)

-trifluoroacetic acid (9470, Baker)

-ammonium sulfate (1.01217, Merck)

-1N sodium hydroxide (1.09137, Merck)

-1N hydrochloric acid (1.09057, Merck)

-hplc grade water (MilliQ)

-water for injection

-anhydrous sodium sulfate (code 6649, Merck)

-methanol (code 06009, Merck)

-Hydrazoic acid,sodium salt (code 6688, Merck)

-biphosphate sodium-hydrate (code 06346, Merck)

-disodium phosphate dihydrate (code 06580, Merck)

Biological test is used:

-Jurkat pKZ142 clones (WCB) 24 cells

-TACI-Fc5(1-8.66mg/ml)

-zTNF4(1.44mg/ml)

-RPMI 1640, contain or do not contain phenol red (Gibco)

-hyclone (FBS) (GIBCO)

-L-glutaminate (Hyclone)

-Sodium Pyruvate (Gibco) puromycin (Sigma)

-stable GLO luciferase test buffer and substrate (ProMegaE2510)

White tissue culture 96 orifice plates are added a cover (Dynex)

-96 orifice plates are added a cover (Falcon)

-5mL polypropylene test tube (Falcon)

Equipment

-HPLC system (Waters)

-metering pipet (Gilson)

-differential scanning calorimeter (2920 types, TA instrument company)

-microcalorimeter (the VP-DSC type, MicroCal)

-pH meter (713 types, Metrohm)

-permeability manometer (Osmomat 030-D, Gonotec)

-optically-active spectrophotometer J-810 is equipped with Peltier temperature control meter PTC-423S (Jasco)

-fluorescence spectrophotometry instrument FluoroMax3 is equipped with microplate counting meter MicroMax384 (Jobin Yvon)

-96-hole MaxSorp plate (Nunc)

-spectrophotometer λ 354 (Perkin-Elmer)

-quartz curette 0.1 and 1cm light path (Perkin Elmer)

-Zetasizer?Nano?Series(Malvern)

-low capacity (about 70 μ L) quartz curette

-cell stirring system (8400 or 8450 types, Amicon)

-10kDa mwco membrane (the YM10 type, Amicon)

-rustless steel container 22mL and 220mL capacity (Sartorius)

-filter membrane 0.22 μ m (Durapore type GWV P, Millipore)

-filter membrane 0.45 μ m (Durapore type HVLP, Millipore)

-fluorescence/RALS photometer (Photon Technology International)

-IKA-Vibrax-VXR agitator (IKA-Works, Inc.)

(Lyoflex 06, and Lyoflex 08, Edwards) for-freeze dryer

-vortex agitator (Falc)

-calorstat (Heraeus)

-cryogenic refrigerator (Angelantoni)

Biological Detection is used:

-luminometer plate reader Lumicount Packard

-Graph Pad Prism software

-laminar flow ventilated chamber (Flow Laboratories)

-37 ℃ of CO ₂Incubator (Heraeus)

-37 ℃ of water baths

-cell counter

-microscope

-shake platform

-desktop centrifuge

-single channel and multichannel graduated pipette and move the liquid head

-move the liquid assistor

Main packaging material

-DIN2R (3mL) vial (Nuova OMPI)

-Flurotec rubber closure (S2F452, D777-1, B2-40, West Pharmaceutical)

-rubber closure (1779W1816 Lycoperdon polymorphum Vitt Pharmagummi)

Method

Molecular size exclusion chromatography (SEC)

Method 1

, get 40 μ l (20 μ g) and be added to tsk gel post G3000SWXL (5 μ m are on 7.8 * 300mm) to 0.5mg/ml with PBS1X pH=7.2 dilute sample.Each test, eluent is the 0.05M sodium acetate, 0.5M ammonium sulfate pH=6.0.

Method 2

, get 40 μ l (20 μ g) and be added to tsk gel post G3000SWXL (5 μ m, on 7.8 * 300mm), this post is connected in tsk gel SWXL guard column (6mm * 4cm) to 0.25mg/ml with the mobile phase dilute sample.Each test, eluent is the 0.05M sodium phosphate, 0.5M ammonium sulfate pH=6.0.

Reverse-phase chromatography (RP)

, get 40 μ l (20 μ g) and be added to and use 71% buffer A (0.1%TFA/ water) and 29% buffer B (0.1%TFA/ acetonitrile) equilibrated to 0.5mg/ml with PBS1X pH=7.2 dilute sample

(8 μ m are on 50 * 4.6mm) for post.With linear gradient liquid elution samples, flow velocity 2ml/ branch.Inject not commensurability titer (IRS ACI-Fc5 2002/2001) and produce standard curve.

The terminal truncate (RP) of C-

Sample with 37 ℃ of digestion of enzyme (Cys-C) 2 hours, (is carried out reversed phase chromatography at the Vydac C18 post of being with guard column then on 4,6 * 50mm).

Eluent A:0.1%TFA/ water; Eluent B:0.08%TFA/70%CH ₃CN

Flow velocity: the 1ml/ branch,

Temperature: 40 °+/-5 ℃,

Detect: 214nm

Gradient: eluent B 15%-23% in 7 minutes, totally 15 minutes.

Pruning form (RP)

Dilute the sample of TACI-Fc medicine to protein concentration 4mg/ml with pure water.The sample of getting 10 μ l dilution is diluted to 200 μ l with the liquid that reduces denaturation (0.15M DTT/6M guanidine hydrochloride), and cultivated 90 minutes for 60 ± 2 ℃ vortex vibration back.Get 75-150 μ l (15-30 μ g) injection and before used initial conditions liquid (71% eluent A, 0.05%TFA/ water and 29% eluent B, 0.04%TFA/ acetonitrile) equilibrated post (macropore butyl post, 5mm, diameter 4.6mm * high 50mm, article No. 7116-05, J.T.Baker company product) in.

Free Fc dimer (IEC)

Sample to protein concentration with Poloxamer188 100mg/L solution (with 10mM sodium phosphate buffer pH4.0 preparation) dilution TACI-Fc medicine is 10mg/ml.When TACI-Fc concentration is higher than 100mg/ml, should carry out diluted sample by weighing.

Get 25 μ l (250 μ g) injection and use initial conditions liquid (80% eluent A in advance; 10mM sodium phosphate pH4.00 and 20% eluent B, 10mM sodium phosphate pH 4.00+0.5M KCI) equilibrated post (ProPac WCX-10G (protection), 4x50mm; article No. 054994, Dionex company product) in.

Oxidised form (MALDI-ToF)

The TACI-Fc drug sample is carried out peptide figure describe, application NALDI-ToF detects quantitative its oxidised form and verifies.

Analytical type super centrifugal (AUC)

Sample is added in the cuvette of two passage carbon tube/epoxy resin crossheads light path 12mm.The condition that as diluent diluted sample is detected with simulation HPLC with the SE-HPLC elution buffer.Corresponding buffer is added in the reference channel (this instrumentation is similar to double beam spectrophotometer).Then the application of sample cuvette is put into the AN-59Ti analytical rotor, be placed in the Beckman Optima XL-I analytical type centrifuge.Adopt following experiment setting to analyze:

Rotary head type: 8 hole rotary heads;

Rotating speed; 40000rpm;

Crosshead: carbon tube/epoxy resin;

Passage length: 12mm;

20 ℃ ± 0.2 ℃ of the temperature of AUC duration of test;

Detect wavelength: 280nm;

Sample concentration: 0.5mg/ml;

Sample volume: 432ml/ passage;

Reference product volume: 442ml/ passage.

With c (s) the method analytical data of the Peter Schuck of NIH exploitation, analyze with its analysis programme SEDFIT (8.7 editions).

Differential scanning calorimetry (DSC)

DSC 2920CE, the TA instrument; Temperature range 25-100 ℃; 2 ℃/minute of programming rates; Maximum volume (HVP) the 75 μ l solution of loading; With placebo solution is reference liquid.Microcalorimeter is MicroCalVP-DSC; Temperature range 25-100 ℃; 70 ℃/hour of programming rates; Reacted=15 seconds; Data pitch 0.2-8 ℃; Specimen cup is loaded the 5mg/ml Taci-Fc5 liquid of about 600ml; Use water as reference solution.

Optical density (OD)

Dilute with water prepares 0.5mg/ml TACI-Fc5 liquid, with the Lambert-(ε is a molar extinction coefficient to Bill's formula OD=ε bc; B is the thickness of light cup) calculating concentration (c).ε(280nm)＝1.56(mL/mg)/cm。These value of calculation be multiply by the concentration that extension rate records initial liquid.

Garden dichromatic (CD)

The structure fate is analysed

Usually adopt CD research peptide and proteinic conformation.Several factors may influence CD spectrum far ultraviolet (180-250nm) district and near ultraviolet band 9250-350nm) form of characteristic peak, as protein concentration, temperature, pH and ionic strength.Reported literature has been represented the secondary structure (alpha-helix, β-lamella curl at random) of particular type in observed total bands of a spectrum position in the far-ultraviolet region.Observed CD bands of a spectrum are mainly produced by Trp, Tyr, Phe and disulfide bond in the near ultraviolet band.

Yet, must be pointed out that the signal of disulfide bond is generally much lower than the signal of aromatic amino acid.As long as these residues are positioned at asymmetric environment and can produce the CD signal.The conformation change of albumen tertiary structure often causes the variation of initial environment, thereby causes that the CD wave spectrum changes.In fact, each aminoacid of native protein occupies the unique location in the three dimensional structure.The change of this structure can cause the change of its accessibility.

Near ultraviolet band CD is spectrographic not to be put:

Scanning speed=5-20nm/ branch; Scope=250-350nm; Response speed=8 second; Concentration=2mg/ml; Optical path length=1cm; Data pitch=0.5nm; Bandwidth=1nm; Accumulation=2 normal sensibilities.Obtain spectrum under the room temperature.

The spectrographic setting of far-ultraviolet region CD;

Scanning speed=5-20nm/ branch; Scope=200-300nm; Response speed=8 second; Concentration=0.25mg/ml; Optical path length 0.1cm; Data pitch=0.5nm; Bandwidth=1nm; Accumulation=2 normal sensibilities.Obtain spectrum under the room temperature.

Protein unfolding temperature

With CD is the valuable method of studying protein secondary and tertiary structure under different temperatures with the scanning of fixed wave length monitor temperature.This detection can be estimated proteinic unfolding temperature (T in different preparations _Unf).Though T _UnfNot directly related with the free energy (it is a kind of index of protein stability) that the protein unfolding discharges, but well accepted be T _UnfRising should increase relevant with protein stability.Therefore, T _UnfVariation can show whether particular composition has stable or go stable effect.Variation by monitoring tryptophan (Trp) signal is studied thermal denaturation, and the variation of this signal varies with temperature relevant with protein conformation.At 55-70 ℃ of scope internal heating medicine preparation (1 ℃/minute).Can pass through of the influence of the change-detection temperature of the ellipse garden of CD relative minimum 292.5nm to tertiary structure.Adopt the 4th grade of multinomial fitting process to calculate the melting temperature value.

The setting of CD temperature scanning

Temperature range=55-70 ℃; Programming rate=1 ℃/minute; λ=292.5nm; Concentration=2mg/ml; Response speed=8 second; Data pitch=0.2-8 ℃; Bandwidth=1nm; Normal sensibility; Mixing speed=low speed.

Dynamic light scattering method (DLS)

Dynamic light scattering method is to detect the light scattering relevant with granular size that the granule Brownian movement produces.This method need place granule under the laser beam, analyzes the strength fluctuation in the scattered light.Or rather, the speed of granule Brownian movement relevant with granular size (Stoker-Einstein's equation).Detect the similarity degree between two kinds of signals (this example is a strength signal) in a period of time with digital correlator, provide the relevant information of nature and extent that fluctuates with scattered light intensity, these information are relevant with granular size.After measuring correlation function, available its calculates particle size distribution.

With the scattered light intensity (anti-phase scattering detection) of Zetasizer Nano Series detection near 180 °.The influence of the influence of multiple scattering light and big contamination particle when this set can reduce by sample.Adopt disposable cuvette (the about 70 μ l of internal capacity).Temperature during detection=25 ℃.Equilibration time=1 minute, number of run=11 time; Running time=10 second; Detect number of times=2; Dispersant is water (viscosity=0.8872cP; Refractive index=1.330), without dilution.

Fluorescence right angle light scattering (RALS)

Detect RALS with fluorescence detector, its exciting light is provided with identical with radiative wavelength.In this kind configuration, fluorescence detector becomes very sensitive RALS detector.RALS raises and shows that cohesion/precipitation is arranged in the sample.

Fluorescence

Interior fluorescence

Protein contains three kinds of aromatic amino acid residues (tryptophan Trp, tyrosine Tyr, phenylalanine Phe), and this becomes its reason of sending interior fluorescence.The fluorescence of folded protein is the set of each aromatic moieties fluorescence.General with 280nm wavelength or longer 295nm optical excitation albumen fluorescence commonly used.Great majority emission light is produced by the trp residue emission, and a few issue light is produced by tyrosine and phenylalanine.The intensity of tryptophan maximum fluorescence emission, quantum rate and wavelength extremely depend on solvent.Along with the reduction of solvent polarity around the trp residue, fluorescence spectrum shifts and is shorter wavelength, and fluorescence intensity increases.The tryptophan that is embedded in the albumen hydrophobic core is compared with the tryptophan of protein surface, its spectrum 10-20nm that may drift about.In addition, tryptophan fluorescence can be by contiguous protonated acid group such as Asp or Glu cancellation.Therefore, can utilize fluorescence, the variation of reflection aromatic moieties microenvironment of living in as potent monitoring tool.

MicroMax384 is a kind of microwell plate readout instrument, can load to reach the dull and stereotyped of 384 holes and connect the FluoroMax spectrofluorophotometer.The exciting light of monochromator and emission light is by fibre bundle turnover MicroMax 384, and therefore, the user can also the measuring exciting light of selection intensity and wavelength of transmitted light be right with this main fluorophotometer scanning.All controls of MicroMax are the automatic control by DataMax software, by this software also can self-defined option board in micropore.

Carry out high throughput fluorescence scanning with the MicroMax384 plate reader, adopt following the setting: exciting light and emission light interval=5nm; λ exc=280nm; Emission optical range=300-450nm; The time of integration=0.1 second; Do not dilute.Automatically calculate the emission maximum optical wavelength with Fluromax3 software.

RALS

Carrying out RALS with FluoroMax spectrofluorophotometer synchronous scanning between 500-800nm (λ exc=λ em) detects.The intensity that shows under these conditions (n.s absorbs and not influenced by light source) is mainly due to (light/protein) scattering phenomenon that exists in the solution.The scattering overall strength increases and increases along with the albumen granularity, and therefore, available this technical monitoring cohesion, subunit separate, degrade or the like.

Scattering strength also depends on protein concentration and dioptric coefficient, therefore, should detect comparison under identical protein concentration.

It is as follows to carry out the parameter that RALS detect to be provided with: synchronous scanning, wave-length coverage=500-800nm, spacing=15nm, the time of integration=0.5 second, skew=0nm, sample concentration=35mg/ml (usefulness milliQ water is as diluent).

The anisotropy of fluorescence emission

Its size, shape and local environment (being solvent) are depended in macromolecular rotation.Usually adopt the polarization emission measurement to come the minor variations (cohesion, combination, cut-out) and the environmental change (local viscosity changes etc. mutually) of detection molecules size.The first step of these detections is to excite selected fluorophor (light selection).Usually absorb the molecular group of dipole with vertical direction excitation energy with orthogonal polarized light.At this moment mutually in, produce vertical polarized excitation light with the polariser in the exciting pathway.In case excited, molecule can be in the excited state lifetime phase (about 10 ^-9Second) internal rotation.This rotation will make the fluorescent emission depolarization.Measure type and degree that the radiative composition of polarization can go out to calculate the molecule rotation.Detect the polarization ingredient of fluorescent emission with the polariser in the emission approach.From the size of vertical (V) and level (H) emission light ingredient, can calculate the degree and the type of rotation.Anisotropy (A) is a kind of ratio, is defined as the overall strength of the intensity of linear polarization ingredient divided by light:

A＝(I _VV-G＊I _VH)/(I _VV+2G＊I _VH)

In this formula, G is a correction factor, G=I _HV/ I _HH

In these equations, first subscript of intensity I is represented the position (H or V) of exciting light polariser, and second is the position (H or V) of emission light polarimeter.When excitation wavelength is made as λ=295nm, the anisotropy of fluorescence provided with trp residue move with they local viscosity of living in for information about.Therefore, the increase of fluorescence anisotropy can reflect that proteinic these residues are in more inflexible environment.

It is as follows to detect the set parameter of anisotropy: λ exc=295nm, emission optical range=500-800nm, the time of integration=0.5 second, spacing=15nm, sample concentration=35mg/ml (with milliQ water as diluent).

Biological test

The test of TACI-Fc extracorporeal biology is based on Jurkat (the acute t cell lymphoma of people) cells transfected (Jurkat pKZ142).With two kinds of these cell lines of plasmid transfection, first kind of total length TACI cDNA that is coded under the control of CMV promoter, second kind contains the luciferase reporter gene that NF-kB/AP-1 drives.This method reacts, causes the activated ability of NF-kB/AP-1 luciferase reporter gene of transfection in conjunction with cell surface taci receptor, triggering signal transductory cascade based on zTNF4.The existence of soluble T ACI-Fc has suppressed combining of zTNF4 and taci receptor, thereby reduces the expression of luciferase.

Jurkat pKZ142 cell is cultivated with the TACI-Fc standard substance, set up the full dose-effect curve of 27.86-1.63U/ml, and cultivate with the given the test agent of two kinds of concentration (promptly 4 and 6U/ml), these two kinds of concentration are positioned at the linear segment of standard curve.

Then zTNF4 solution is added in standard substance and the sample with the concentration (being every hole 150ng/ml) that can induce the generation of secondary maximum luciferase; Be produced as contrast with minimum and maximum luciferases.37 ℃ of (5%CO ₂) cultivate after 4 hours, add cell with luciferase Steady Glo test kit, detect the expression of luciferase with luminous photometer.

Y value (RLU) in two kinds of test concentrations of linear segment interpolation of standard dose response curve calculates sample usefulness, obtains the TACI-Fc concentration (Graph Pad software) on the X-axis.Try to achieve the meansigma methods of two kinds of concentration in each time independent trials, the usefulness arithmetic mean of instantaneous value that obtains from each time independent trials calculates the biologic activity of TACI-Fc5 then.

The preformulation process

PH, buffer type and adjuvant are estimated the influence of protein stability.The TACI-Fc solution of preparation concentration 70 or 100mg/ml, the following variable of preliminary study:

-pH(4、5、6、7)

-buffer (acetate, phosphate, succinate, citrate, histidine)

-sugar (mannitol, sorbitol, glycerol, sucrose, trehalose)

-adjuvant (sodium chloride, magnesium chloride, calcium chloride, glycine)

In addition, the TACI-Fc5 to 70mg/ml has carried out the research of following further prescreen:

-carry out multigelation (F-T) 1,3,5F-T time with the 20mM buffer (histidine, phosphate, succinate, citrate) of pH 5-6-7;

-40 ℃ of cultivations (shake and do not shake);

-being stored in 2-8 ℃, pH 4-5-6 is in the 20mM buffer (acetate, histidine, phosphate).

According to these first observed result, select two kinds of buffer (phosphate and histidine) of pH 5 and 6, prepare second batch of preparation, in order to study the effect that adds stabilizing agent (residuals weight mole osmotic concentration 0.280 OSM) again.Tested following stabilizing agent: glucose, mannitol, sorbitol, trehalose, glycine, NaCl, MgCl ₂, CaCl ₂

Each solution is stored in 2-8 ℃, 25 ℃ and 40 ℃, in 14 days, condensation product (SE-HPLC), protein content (RP-HPLC), pH and outward appearance is detected.

Experimental design

According to the selection done in early stage, the design that experimentizes is with the influence to protein stability of the factor of the varying level studied before assessing.Preparation in acetate and the histidine buffering liquid is detected, detect simultaneously following table surface-active agent: Poloxamer 188 ( F-68) and polysorbas20, and following adjuvant: arginine, glycine, lysine, mannitol and trehalose.These preparations are contained in the vial, are stored in 2-8 ℃, 25 ℃ and 40 ℃, detect condensation product (SE-HPLC and AUC), pH, outward appearance and osmolality.Also adopt the biological and physical analysis method (as garden dichromatic, 2 ^NdUV derive spectrographic method, interior fluorescence method).

Candidate's preparation

When the pre-preparation phase finishes, detect and to have identified some candidate's preparation, contain 70 or 100mg/mlTACI-Fc, 10mM acetate buffer, mannitol (51mg/ml) or anhydrous trehalose (80 or 96mg/ml) for adjuvant have or do not have Poloxamer 188 (

F-68) (0.05mg/ml), pH4.8,5.0,5.2 and 5.4.

All solution are collected in the sterilization container after by 0.22 μ m Durapore membrane filtration degerming.Then solution is loaded in the DIN2R vial (the 1ml amount of filling out).Get processed sample (filtering forward and backward) loss of evaluating protein matter or increasing of condensation product during the preparation.

Sample is stored in 2-8 ℃, 25 ℃ and 40 ℃, detects the result who stores 1 month (40 ℃) and 6 months (2-8 ℃ and 25 ℃).

Detect condensation product (SE-HPLC), protein content (RP-HPLC), pH, osmolality and the biologic activity of candidate's preparation.Also detect the degree of the terminal truncate of C-and the ratio of truncate/pruning form.Also adopted bio-physical method (interior fluorescence, dynamic light scattering, 90 ° of light scattering).

Also assessed the influence of freeze-thaw candidate's preparation liquid sample to 2-8 ℃ of preservation :-80 ℃ of freezing samples room temperature then melt.Assess the condensation product content of freeze-thaw front and back with SE-HPLC.

Estimated simulation medicine shipment transportation and shaken 24 hours 2-8 ℃ of influence of preserving sample, with sample be placed on microwell plate shake room temperature on the platform shake after 24 hours with SE-HPLC assessment condensation product level and with originally level relatively.

The result

Candidate's preparation is prepared with 10mM NaAc.

Following table A-T has reported detailed results.

Total condensation product percentage ratio % (2-8 ℃) that Table A: SE-HPLC measures

	??TACI-Fc5??(mg/ml)	Form	Time 0	4 weeks	6 weeks	8 weeks	12 weeks	16 weeks	26 weeks
										??21A	??70	??pH?5，96mg/ml?Tre，F680.05	??3,3	??3,6	??3,7	??3,2	??2,9	??-	??2,0
??21B	??70	??pH?5，80mg/ml?Tre	??3,4	??3,7	??3,8	??3,5	??3,0	??-	??2,1
										??21C	??70	??pH?5.4，80mg/ml?Tre，F680.05	??3,4	??3,6	??3,8	??3,9	??3,1	??-	??2,2
??21D	??100	??pH?5，80mg/ml?Tre，F680.05	??3,4	??3,6	??3,9	??3,5	??3,0	??-	??2,3
										??21E	??100	??pH?5，80mg/ml?Tre	??3,7	??4,5	??3,8	??3,8	??3,3	??-	??2,3
??21F	??100	??pH?5.4，80mg/ml?Tre，F680.05	??3,6	??3,7	??3,9	??3,4	??3,4	??-	??2,8
										??21G	??100	??pH?5，51mg/ml?Man，F680.05	??3,7	??3,7	??3,9	??4,1	??3,2	??-	??2,6
??21H	??100	??pH?5，51mg/ml?Man	??3,8	??3,7	??4,1	??3,6	??3,3	??-	??2,6
										??21I	??100	??pH?4.8，80mg/ml?Tre，F680.05	??3,6	??3,3	??2,9	??3,0	??-	??2,6	??-
??21L	??100	??pH?5.2，80mg/ml?Tre，F680.05	??3,6	??3,4	??3,3	??3,3	??-	??2,7	??-

Used batch materials, and S128/L20a (the 10mM sodium acetate buffer, pH=5.0), total condensation product %=3.9 (Tre=trehalose, Man=mannitol).

Total condensation product percentage ratio % (25 ℃) that table B SE-HPLC measures

	??TACI-Fc5??(mg/ml)	Form	Time 0	2 weeks	4 weeks	6 weeks	8 weeks	12 weeks	17 weeks	27 weeks
											??21A	??70	??pH?5，96mg/ml?Tre，F680.05	??3,3	??2,6	??4,0	??3,8	??3,7	??3,5	??-	??3,4
??21B	??70	??pH?5，80mg/ml?Tre	??3,4	??3,0	??3,7	??4,1	??3,9	??3,4	??-	??3,5
											??21C	??70	??pH?5.4，80mg/ml?Tre，F680.05	??3,4	??3,1	??3,9	??4,2	??4,6	??4,0	??-	??4,5
??21D	??100	??pH?5，80mg/ml?Tre，F680.05	??3,4	??5,0	??4,0	??4,1	??4,4	??4,2	??-	??4,4
											??21E	??100	??pH?5，80mg/ml?Tre	??3,7	??3,3	??3,9	??4,6	??4,5	??4,4	??-	??4,7
??21F	??100	??pH?5.4，80mg/ml?Tre，F680.05	??3,6	??3,8	??4,5	??5,0	??5,1	??5,2	??-	??6,5
											??21G	??100	??pH?5，51mg/ml?Man，F680.05	??3,7	??3,9	??3,9	??4,7	??4,7	??4,5	??-	??4,9
??21H	??100	??pH?5，51mg/ml?Man	??3,8	??3,4	??3,9	??4,6	??4,5	??4,4	??-	??4,9
											??21I	??100	??pH?4.8，80mg/ml?Tre，F680.05	??3,6	??3,0	??3,6	??3,3	??3,5	??-	??5,0	??-
??21L	??100	??pH?5.2，80mg/ml?Tre，F680.05	??3,6	??3,4	??4,1	??3,8	??4,2	??-	??4,9	??-

Used batch materials, and S128/L20a (the 10mM sodium acetate buffer, pH=5.0), total condensation product %=3.9 (Tre=trehalose, Man=mannitol).

Total condensation product percentage ratio % (40 ℃) that table C.SE-HPLC measures

	??TACI-Fc5??(mg/ml)	Form	Time 0	1 week	2 weeks	4 weeks
							??21A	??70	??pH?5，96mg/ml?Tre，F680.05	??3,3	??3,9	??4,1	??6,1
??21B	??70	??pH?5，80mg/ml?Tre	??3,4	??4,0	??4,4	??6,3
							??21C	??70	??pH?5.4，80mg/ml?Tre，F680.05	??3,4	??4,4	??5,3	??7,4
??21D	??100	??pH?5，80mg/ml?Tre，F680.05	??3,4	??5,0	??5,3	??7,9
							??21E	??100	??pH?5，80mg/ml?Tre	??3,7	??5,0	??5,5	??7,9
??21F	??100	??pH?5.4，80mg/ml?Tre，F680.05	??3,6	??6,2	??7,4	??11,0
							??21G	??100	??pH?5，51mg/ml?Man，F680.05	??3,7	??5,0	??6,1	??8,4
??21H	??100	??pH?5，51mg/ml?Man	??3,8	??5,9	??6,1	??8,4
							??21I	??100	??pH?4.8，80mg/ml?Tre，F680.05	??3,6	??6,1	??7,4	??12,2
??21L	??100	??pH?5.2，80mg/ml?Tre，F680.05	??3,6	??7,6	??8,9	??14,0

Used batch materials, and S128/L20a (the 10mM sodium acetate buffer, pH=5.0), total condensation product %=3.9 (Tre=trehalose, Man=mannitol).

The dimer percentage ratio % (2-8 ℃) that table D.AUC measures

	??TACI-Fc5??(mg/ml)	Form	4 weeks	8 weeks	13 weeks
						??21A	??70	??pH?5，96mg/ml?Tre，F680.05	??4,0	??7,4	??3,0
??21B	??70	??pH?5，80mg/ml?Tre	??3,2	??1,6	??3,6
						??21C	??70	??pH?5.4，80mg/ml?Tre，F680.05	??-	??7,5	??2,6
??21D	??100	??pH?5，80mg/ml?Tre，F680.05	??-	??4,2	??3,7
						??21E	??100	??pH?5，80mg/ml?Tre	??3,3	??4,5	??3,2
??21F	??100	??pH?5.4，80mg/ml?Tre，F680.05	??4,0	??2,9	??4,1
						??21G	??100	??pH?5，51mg/ml?Man，F680.05	??3,1	??4,0	??3,1
??21H	??100	??pH?5，51mg/ml?Man	??3,7	??4,7	??2,8

	??TACI-Fc5??(mg/ml)	Form	4 weeks	8 weeks	13 weeks
						??21I	??100	??pH?4.8，80mg/ml?Tre，F680.05	??3,9	??-	??-
??21L	??100	??pH?5.2，80mg/ml?Tre，F680.05	??3,5	??-	??-

Table E.AUC measures the big condensation product percentage ratio % (2-8 ℃) that obtains

	??TACI-Fc5??(mg/ml)	Form	4 weeks	8 weeks	13 weeks
						??21A	??70	??pH?5，96mg/ml?Tre，F680.05	??2,3	??2,8	??1,1
??21B	??70	??pH?5，80mg/ml?Tre	??2,0	??0,5	??2,5
						??21C	??70	??pH?5.4，80mg/ml?Tre，F680.05	??-	??3.0	??0,6
??21D	??100	??pH?5，80mg/ml?Tre，F680.05	??-	??2,1	??1,4
						??21E	??100	??pH?5，80mg/ml?Tre	??1,2	??1,5	??1,2
??21F	??100	??pH?5.4，80mg/ml?Tre，F680.05	??1,1	??0,8	??1,6
						??21G	??100	??pH?5，51mg/ml?Man，F680.05	??1,5	??2,1	??1,5
??21H	??100	??pH?5，51mg/ml?Man	??1,9	??2,1	??1,3
						??21I	??100	??pH?4.8，80mg/ml?Tre，F680.05	??1,9	??-	??-
??21L	??100	??pH?5.2，80mg/ml?Tre，F680.05	??2,0	??-	??-

Table F.AUC measures the dimer percentage ratio % (25 ℃) that obtains

	??TACI-Fc5??(mg/ml)	Form	T=0 (5 ℃ of 4 weeks)	4 weeks	8 weeks	13 weeks
							??21A	??70	??pH?5，96mg/ml?Tre，F680.05	??4,0	??2,6	??3,8	??5,3
??21B	??70	??pH?5，80mg/ml?Tre	??3,2	??3,9	??3,4	??2,7
							??21C	??70	??pH?5.4，80mg/m?l?Tre，F680.05	??-	??2,8	??4,0	??5,6
??21D	??100	??pH?5，80mg/ml?Tre，F680.05	??-	??2,4	??4,2	??3,2
							??21E	??100	??pH?5，80mg/ml?Tre	??3,3	??3,3	??3,7	??3,7
??21F	??100	??pH?5.4，80mg/ml?Tre，F680.05	??4,0	??4,3	??5,2	??4,2
							??21G	??100	??pH?5，51mg/ml?Man，F680.05	??3,1	??3,9	??3,9	??3,8
??21H	??100	??pH?5，51mg/ml?Man	??3,7	??3,0	??4,3	??6,6
							??21I	??100	??pH?4.8，80mg/ml?Tre，F680.05	??3,9	??-	??-	??-
??21L	??100	??pH?5.2，80mg/ml?Tre，F680.05	??3,5	??-	??-	??-

Table G.AUC measures the big condensation product percentage ratio % (25 ℃) that obtains

	??TACI-Fc5??(mg/ml)	Form	T=0 (5 ℃ of 4 weeks)	4 weeks	8 weeks	13 weeks
							??21A	??70	??pH?5，96mg/ml?Tre，F680.05	??2,3	??0,3	??0,8	??2,5
??21B	??70	??pH?5，80mg/ml?Tre	??2,0	??1,1	??0,6	??0,2
							??21C	??70	??pH?5.4，80mg/ml?Tre，F680.05	??-	??0,1	??0,8	??1,3
??21D	??100	??pH?5，80mg/ml?Tre，F680.05	??-	??0,4	??1,3	??0,2
							??21E	??100	??pH?5，80mg/ml?Tre	??1,2	??0,9	??0,9	??0,9
??21F	??100	??pH?5.4，80mg/ml?Tre，F680.05	??1,1	??1,6	??1,2	??0,7
							??21G	??100	??pH?5，51mg/ml?Man，F680.05	??1,5	??1,9	??1,1	??1,1
??21H	??100	??pH?5，51mg/ml?Man	??1,9	??1,5	??2,6	??3,6

	??TACI-Fc5??(mg/ml)	Form	T=0 (5 ℃ of 4 weeks)	4 weeks	8 weeks	13 weeks
							??21I	??100	??pH?4.8，80mg/ml?Tre，F680.05	??1,9	??-	??-	??-
??21L	??100	??pH?5.2，80mg/ml?Tre，F680.05	??2,0	??-	??-	??-

Table H.AUC measures the two condensation product percentage ratio % (40 ℃) that obtain

	??TACI-Fc5??(mg/ml)	Form	T=0 (5 ℃ of 4 weeks)	2 weeks	3 weeks
						??21A	??70	??pH?5，96mg/ml?Tre，F680.05	??4,0	??-	??4,4
??21B	??70	??pH?5，80mg/ml?Tre	??3,2	??-	??3,9
						??21C	??70	??pH?5.4，80mg/ml?Tre，F680.05	??-	??-	??5,3
??21D	??100	??pH?5，80mg/ml?Tre，F680.05	??-	??-	??-
						??21E	??100	??pH?5，80mg/ml?Tre	??3,3	??-	??6,4
??21F	??100	??pH?5.4，80mg/ml?Tre，F680.05	??4,0	??-	??7,0
						??21G	??100	??pH?5，51mg/ml?Man，F680.05	??3,1	??-	??6,0
??21H	??100	??pH?5，51mg/ml?Man	??3,7	??-	??6,4
						??21I	??100	??pH?4.8，80mg/ml?Tre，F680.05	??3,9	??4,9	??-
??21L	??100	??pH?5.2，80mg/ml?Tre，F680.05	??3,5	??8,0	??-

Table I .AUC measures the big condensation product percentage ratio % (40 ℃) that obtains

	??TACI-Fc5??(mg/ml)	Form	T=0 (5 ℃ of 4 weeks)	2 weeks	3 weeks
						??21A	??70	??pH?5，96mg/ml?Tre，F680.05	??2,3	??-	??1,8
??21B	??70	??pH?5，80mg/ml?Tre	??2,0	??-	??1,5
						??21C	??70	??pH?5.4，80mg/ml?Tre，F680.05	??-	??-	??2,0
??21D	??100	??pH?5，80mg/ml?Tre，F680.05	??-	??-	??-
						??21E	??100	??pH?5，80mg/ml?Tre	??1,2	??-	??2,8
??21F	??100	??pH?5.4，80mg/ml?Tre，F680.05	??1,1	??-	??2,6
						??21G	??100	??pH?5，51mg/ml?Man，F680.05	??1,5	??-	??3,0
??21H	??100	??pH?5，51mg/ml?Man	??1,9	??-	??3,9
						??21I	??100	??pH?4.8，80mg/ml?Tre，F680.05	??1,9	??5,8	??-
??21L	??100	??pH?5.2，80mg/ml?Tre，F680.05	??2,0	??4,6	??-

Table J.SE-HPLC measures the protein content (2-8 ℃) that obtains

	??TACI-Fc5??(mg/ml)	Form	Time 0	4 weeks	6 weeks	8 weeks	12 weeks	16 weeks	26 weeks
										??21A	??70	??pH?5，96mg/ml?Tre，F680.05	??64,7	??64,2	??66,3	??63,2	??66,9		??66,0
??21B	??70	??pH?5，80mg/ml?Tre	??69,8	??62,2	??67,3	??62,1	??67,6		??63,1
										??21C	??70	??pH?5.4，80mg/ml?Tre，F680.05	??62,1	??64,7	??66,4	??63,7	??65,7		??64,4
??21D	??100	??pH?5，80mg/ml?Tre，F680.05	??90,7	??91,5	??91,8	??89,1	??96,8		??92,5
										??21E	??100	??pH?5，80mg/ml?Tre	??92,6	??97,7	??99,8	??87,7	??92,9		??92,4
??21F	??100	??pH?5.4，80mg/ml?Tre，F680.05	??96,1	??91,8	??94,3	??90,4	??92,8		??93,5
										??21G	??100	??pH?5，51mg/ml?Man，F680.05	??94,7	??100,4	??95,7	??91,2	??93,3		??90,4
??21H	??100	??pH?5，51mg/ml?Man	??89,0	??98,6	??90,6	??89,0	??93,3		??88,8
										??21I	??100	??pH?4.8，80mg/ml?Tre，F680.05	??94,3	??99,5	??88,8	??100,0		??91,5
??21L	??100	??pH?5.2，80mg/ml?Tre，F680.05	??96,9	??85,2	??93,3	??99,6		??98,2

Table K.SE-HPLC measures the protein content (25 ℃) that obtains

	??TACI-Fc5??(mg/ml)	Form	Time 0	2 weeks	4 weeks	6 weeks	8 weeks	12 weeks	17 weeks	27 weeks
											??21A	??70	??pH?5，96mg/ml?Tre，F680.05	??64,7	??61,6	??66,8	??67,5	??65,3	??68,3		??63,3
??21B	??70	??pH?5，80mg/ml?Tre	??69,8	??60,0	??63,2	??66,1	??63,4	??64,7		??62,7
											??21C	??70	??pH?5.4，80mg/ml?Tre，F680.05	??62,1	??59,0	??64,1	??66,8	??64,5	??64,4		??62,9
??21D	??100	??pH?5，80mg/ml?Tre，F680.05	??90,7	??99,5	??89,6	??94,9	??91,6	??94,3		??89,4
											??21E	??100	??pH?5，80mg/ml?Tre	??92,6	??85,0	??91,0	??98,0	??92,0	??95,6		??82,1
??21F	??100	??pH?5.4，80mg/ml?Tre，F680.05	??96,1	??n.v.	??90,9	??96,0	??94,5	??94,4		??91,1
											??21G	??100	??pH?5，51mg/ml?Man，F680.05	??94,7	??88,4	??96,1	??92,7	??105,0	??93,5		??87,8
??21H	??100	??pH?5，51mg/ml?Man	??89,0	??88,4	??94,4	??94,7	??89,9	??91,3		??88,5
											??21I	??100	??pH?4.8，80mg/ml?Tre，F680.05	??94,3	??95,3	??97,8	??89,7	??98,6		??95,2
??21L	??100	??pH?5.2，80mg/ml?Tre，F680.05	??96,9	??100,0	??100,1	??92,7	??103,0		??92,7

Table L.SE-HPLC measures the protein content (40 ℃) that obtains

	??TACI-Fc5??(mg/ml)	Form	Time 0	??1w	2 weeks	4 weeks
							??21A	??70	??pH?5，96mg/ml?Tre，F680.05	??64,7	??64,9	??60,9	??67,1
??21B	??70	??pH?5，80mg/ml?Tre	??69,8	??64,5	??59,8	??66,9
							??21C	??70	??pH?5.4，80mg/ml?Tre，F680.05	??62,1	??63,8	??61,2	??65,2
??21D	??100	??pH?5，80mg/ml?Tre，F680.05	??90,7	??89,3	??85,7	??91,4
							??21E	??100	??pH?5，80mg/ml?Tre	??92,6	??90,5	??86,2	??88,6
??21F	??100	??pH?5.4，80mg/ml?Tre，F680.05	??96,1	??93,5	??85,1	??90,7
							??21G	??100	??pH?5，51mg/ml?Man，F680.05	??94,7	??88,6	??85,6	??97,8
??21H	??100	??pH?5，51mg/ml?Man	??89,0	??93,0	??83,9	??96,5
							??21I	??100	??pH?4.8，80mg/ml?Tre，F680.05	??94,3	??92,0	??91,2	??99,7
??21L	??100	??pH?5.2，80mg/ml?Tre，F680.05	??96,9	??97,1	??94,9	??103,3

Table M.pH value (2-8 ℃)

	??TACI-Fc5??(mg/ml)	Form	Time 0	4 weeks	6 weeks	8 weeks	12 weeks	16 weeks	26 weeks
										??21A	??70	??pH?5，96mg/ml?Tre，F680.05	??5,2	??5,1	??5,1	??5,1	??5,1	??-	??5,1
??21B	??70	??pH?5，80mg/ml?Tre	??5,2	??5,1	??5,1	??5,1	??5,1	??-	??5,1
										??21C	??70	??pH?5.4，80mg/ml?Tre，F680.05	??5,4	??5,3	??5,3	??5,3	??5,3	??-	??5,3
??21D	??100	??pH?5，80mg/ml?Tre，F680.05	??5,1	??5,0	??5,0	??5,0	??5,0	??-	??5,0
										??21E	??100	??pH?5，80mg/ml?Tre	??5,1	??5,0	??5,1	??5,0	??5,1	??-	??5,1
??21F	??100	??pH?5.4，80mg/ml?Tre，F680.05	??5,5	??5,4	??5,4	??5,4	??5,3	??-	??5,4
										??21G	??100	??pH?5，51mg/ml?Man，F680.05	??5,2	??5,1	??5,1	??5,1	??5,1	??-	??5,1
??21H	??100	??pH?5，51mg/ml?Man	??5,2	??5,1	??5,1	??5,1	??5,1	??-	??5,1
										??21I	??100	??pH?4.8，80mg/ml?Tre，F680.05	??4,8	??4,8	??4,9	??4,8	??-	??4,8	??-
??21L	??100	??pH?5.2，80mg/ml?Tre，F680.05	??5,2	??5,2	??5,3	??5,2	??-	??5,2	??-

Table N.pH value (25 ℃)

	??TACI-Fc5??(mg/ml)	Form	Time 0	2 weeks	4 weeks	6 weeks	8 weeks	12 weeks	17 weeks	27 weeks
											??21A	??70	??pH?5，96mg/ml?Tre，F680.05	??5,2	??5,2	??5,1	??5,1	??5,0	??5,1	??-	??5,1
??21B	??70	??pH?5，80mg/ml?Tre	??5,2	??5,1	??5,0	??5,1	??5,0	??5,1	??-	??5,1
											??21C	??70	??pH?5.4，80mg/ml?Tre，F680.05	??5,4	??5,4	??5,3	??5,3	??5,3	??5,4	??-	??5,3
??21D	??100	??pH?5，80mg/ml?Tre，F680.05	??5,1	??5,0	??5,0	??5,0	??5,1	??5,1	??-	??5,0
											??21E	??100	??pH?5，80mg/ml?Tre	??5,1	??5,1	??5,0	??5,1	??5,0	??5,1	??-	??5,0
??21F	??100	??pH?5.4，80mg/ml?Tre，F680.05	??5,5	??5,4	??5,4	??5,4	??5,4	??5,4	??-	??5,3
											??21G	??100	??pH?5，51mg/ml?Man，F680.05	??5,2	??5,1	??5,1	??5,1	??5,1	??5,1	??-	??5,1
??21H	??100	??pH?5，51mg/ml?Man	??5,2	??5,1	??5,1	??5,1	??5,1	??5,1	??-	??5,1
											??21I	??100	??pH?4.8，80mg/ml?Tre，F680.05	??4,8	??4,8	??4,8	??4,9	??4,9	??-	??4,9	??-
??21L	??100	??pH?5.2，80mg/ml?Tre，F680.05	??5,2	??5,3	??5,2	??5,2	??5,3	??-	??5,2	??-

Table O.pH value (40 ℃)

	??TACI-Fc5??(mg/ml)	Form	Time 0	1 week	2 weeks	4 weeks
							??21A	??70	??pH?5，96mg/ml?Tre，F680.05	??5,2	??5,1	??5,2	??5,1
??21B	??70	??pH?5，80mg/ml?Tre	??5,2	??5,1	??5,1	??5,1
							??21C	??70	??pH?5.4，80mg/ml?Tre，F680.05	??5,4	??5,4	??5,4	??5,4
??21D	??100	??pH?5，80mg/ml?Tre，F680.05	??5,1	??5,0	??5,1	??5,0
							??21E	??100	??pH?5，80mg/ml?Tre	??5,1	??5,1	??5,1	??5,0
??21F	??100	??pH?5.4，80mg/ml?Tre，F680.05	??5,5	??5,4	??5,4	??5,4
							??21G	??100	??pH?5，51mg/ml?Man，F680.05	??5,2	??5,1	??5,1	??5,1
??21H	??100	??pH?5，51mg/ml?Man	??5,2	??5,1	??5,1	??5,1
							??21I	??100	??pH?4.8，80mg/ml?Tre，F680.05	??4,8	??4,9	??4,9	??4,9
??21L	??100	??pH?5.2，80mg/ml?Tre，F680.05	??5,2	??5,3	??5,3	??5,3

Table P. osmolality (OSM/kg)

	??TACI-Fc5??(mg/ml)	Form	??T＝0
				??21A	??70	??pH?5，96mg/ml?Tre，F680.05	??0,444
??21B	??70	??pH?5，80mg/ml?Tre	??0,359
				??21C	??70	??pH?5.4，80mg/m?l?Tre，F680.05	??0,359
??21D	??100	??pH?5，80mg/ml?Tre，F680.05	??0,409
				??21E	??100	??pH?5，80mg/ml?Tre	??0,414
??21F	??100	??pH?5.4，80mg/ml?Tre，F680.05	??0,414
				??21G	??100	??pH?5，51mg/ml?Man，F680.05	??0,445

	??TACI-Fc5??(mg/ml)	Form	??T＝0
				??21H	??100	??pH?5，51mg/ml?Man	??0,438
??21I	??100	??pH?4.8，80mg/ml?Tre，F680.05	??0,388
				??21L	??100	??pH?5.2，80mg/ml?Tre，F680.05	??0,368

Table Q. biological test (U/ml) (2-8 ℃)

	??TACI-Fc5??(mg/ml)	Form	Expected value	Time 0	4 weeks	8 weeks	??13w
								??21A	??70	??pH?5，96mg/ml?Tre，F680.05	??350000	??347704	??365778	??319050	??387896
??21B	??70	??pH?5，80mg/ml?Tre	??350000	??336185	??318012	??289824	??357861
								??21C	??70	??pH?5.4，80mg/ml?Tre，F680.05	??350000	??323222	??333715	??306941	??335181
??21D	??100	??pH?5，80mg/ml?Tre，F680.05	??500000	??461204	??452442	??431738	??489743
								??21E	??100	??pH?5，80mg/ml?Tre	??500000	??458617	??439084	??435680	??470251
??21F	??100	??pH?5.4，80mg/ml?Tre，F680.05	??500000	??455676	??470037	??407200	??424278
								??21G	??100	??pH?5，51mg/ml?Man，F680.05	??500000	??494293	??543338	??401277	??445267
??21H	??100	??pH?5，51mg/ml?Man	??500000	??471667	??446056	??387503	??445371

Table R. biological test (U/ml) (25 ℃)

	??TACI-Fc5??(mg/ml)	Form	Expected value	Time 0	4 weeks	8 weeks	??13w
								??21A	??70	??pH?5，96mg/ml?Tre，F680.05	??350000	??347704	??311541	??279159	??304644
??21B	??70	??pH?5，80mg/ml?Tre	??350000	??336185	??302066	??268469	??342827
								??21C	??70	??pH?5.4，80mg/ml?Tre，F680.05	??350000	??323222	??315938	??269441	??318150
??21D	??100	??pH?5，80mg/ml?Tre，F680.05	??500000	??461204	??508830	??431335	??418713
								??21E	??100	??pH?5，80mg/ml?Tre	??500000	??458617	??441768	??396159	??405400
??21F	??100	??pH?5.4，80mg/ml?Tre，F680.05	??500000	??455676	??449141	??387455	??476384
								??21G	??100	??pH?5，51mg/ml?Man，F680.05	??500000	??494293	??496542	??433228	??440685
??21H	??100	??pH?5，51mg/ml?Man	??500000	??471667	??465114	??365910	??433696

The terminal truncate of table S.C-

Sample	Stabilization time	Truncate
			S128/L20a in batches	??-	??95.0％

Sample	Stabilization time	Truncate
			??21E	8 months 2-8 ℃	??95.3％
??21E	8 months 25 ℃	??94.7％

Table T. prunes form

Sample	Stabilization time	Peak 1 (Fc fragment)	Peak 2+3	Peak 4 (complete)	The total pruning
						S128/L20a in batches	??-	??4.2％	??14.0％	??81.8％	??18.2％
??21E	10 months 2-8 ℃	??4.5％	??15.0％	??80.5％	??19.5％
						??21E	10 months 25 ℃	??8.2％	??37.5％	??54.3％	??45.7％

Main result sums up

The molecular size exclusion chromatography

At 25 ℃, the 70mg/ml preparation is with regard to the cohesion rate, and its slope (the % cohesion/moon) is lower than the 100mg/ml group usually.In one group, the slope that preparation 21D and 21E show is minimum in the back.In the time of 40 ℃, in 100mg/ml liquid preparation group, the slope value that preparation 21E shows is minimum.

AUC

At 2-8 ℃ and 25 ℃, the AUC state of not observing preparation 21E changes to some extent, and the monomer tendency increases in (research) and the 70mg/ml preparation detects between whole stable phase.

Unfolding with the CD monitoring

In 100mg/ml preparation group, preparation 21E shows the highest Tunf, and pH value is different from and causes low T _Unf5.0.The 70mg/ml preparation then shows higher T _UnfValue (promptly stability better).

Interior fluorescence

At 40 ℃, detecting the emission maximum optical wavelength that each preparation of 70mg/ml and 100mg/ml organize alternative preparation 21E has minor variations.

RALS

After 40 ℃ of storages, its RALS of preparation that pH is different from " optimum pH " 5.0 has significantly and increases.The scattering light value that contains mannitol formulations is higher than other preparation.Preparation 21A, 21B, 21D and 21E are the preparations that shows low scattering light value.After the 2-8 ℃ of storage, they do not show the increase of any scattered light.

The anisotropy of fluorescence emission

Preparation 21A and 21B do not observe anisotropic variation after 40 ℃ of storages (1 month).Preparation 21F has relevant change with 21H, and other preparation is observed the variation that is between the two.

Dynamic light scattering

Do not detect size distribution at 2-8 ℃ relevant the change arranged.Observing larger particles after 25 ℃ of storages reduces to some extent.After 40 ℃ of storages, except the preparation that contains mannitol and pH were different from 5.0 preparation, the higher material of molecular weight did not have remarkable increase in all preparations.

The free energy of unfolding

In the 100mg/ml preparation group, preparation 21D and 21E observe higher thermodynamic stability.

Biological test

Store for 25 ℃ and 40 ℃ and do not see that biologic activity reduced in 3 months.

Overall conclusion

Prescreen to liquid preparation studies show that making the solution-stabilized best pH of 70mg/ml TACI-Fc5 is about 5.0.PH value is high more, and it is strong more coacervation (estimating with SE-HPLC) and lowering of concentration (estimating with RP-HPLC and optical density) to occur.Existing of salt (as NaCl, CaCl ₂And MgCl ₂) also cause cohesion to increase.As shown in the two color conformation researchs of the garden of (pH=4.0 compares with 5.0) in the different buffer, being lower than 5 pH value is not optimum pH.The DSC preliminary study shows that trehalose and sucrose have certain positive influences to stability of molecule when unfolding temperature (promptly higher).

Experimental design stage purpose be several adjuvants that research is dissolved in pH=5.0 acetate or histidine buffering liquid (also testing different buffer intensities) exist surfactant as

Effect when F-68 and polysorbas20.The acetate buffer of low concentration provides higher anti-stability to degradation for sample when having mannitol or sorbitol.On stable protein,

F-68 it seems more effective than polysorbas20.

Fluorescence and dynamic light scattering detect consistent with these results.

TACI-Fc candidate sample with bench scale preparation 70mg/ml and 100mg/ml concentration.Make adjuvant (having sodium acetate buffer, pH=4.8,5.0,5.2 and 5.4) with trehalose and mannose.With SE-HPLC and AUC and several spectrophotography monitoring candidate preparation over time.

This result of study is that the low more cohesion that causes of protein concentration is also few more.Confirm that best pH is 5.0.For stablizing the TACI-Fc preparation, trehalose is more successful than mannitol.In 100mg/mlTACI-Fc candidate preparation, preparation 21E (10mM NaAc, pH=5.0,80mg/ml anhydrous trehalose) has shown 40 ℃ of resistances (cohesion of 2-8 ℃ of detection does not have relevant the increasing of statistics) that cohesion is stronger.Or rather, 2-8 ℃ of candidate's liquid preparation 21E purity in 26 weeks improves 1.4%.Its purity only reduced for 1% (26 week) in the time of 25 ℃.

With the RP-HPLC analyzing and testing whole pruning forms of candidate's preparation 21E (2-8 ℃ and 25 ℃, 10 months), compare with initiation material medicine (about 19%), prune the content no change of form.Find that the terminal truncate of C-is about 95%, similar to the initiation material medicine, the truncate of this level is common in people's antibody.

Also analyzed the content of oxidised form among the liquid candidate preparation 21E (storing 10 months for 2-8 ℃ and 25 ℃), compared, do not seen the remarkable change (about 2.4%) of oxidised form storage period with the basic material medicine of this preparation of preparation with RP-MALDI.

The compatibility of embodiment 2:TACI-Fc and antibacterial

Purpose

The purpose of this research is the compatibility of TACI-Fc and different antibacterial in the assessment multiple dose preparation.Detected following antibacterial: 0.9% benzyl alcohol, 0.3% metacresol, 0.5% phenol, 0.5% chlorobutanol, 0.5% phenylethanol, 0.3% benzyl alcohol+0.001% benzalkonium chloride.

Main result

Medicine+antibacterial:

-there is the medicine of antiseptic existence to compare with reference sample (not adding antibacterial), after 40 ℃ of storages deposited for 2 weeks, observe total condensation product (using SEC) and increase (15-70%); It is comparable to observe degradation rate when 25 ℃ and 2-8 ℃.

-antibacterial of minimum negative effect (analyzing according to SEC and CD) is provided is benzyl alcohol+benzalkonium chloride.

Liquid candidate preparation+antibacterial:

-add the cohesion (40 ℃, the 2 week about 10-25% in back) that cohesion that antiseptic causes significantly is lower than medicine and is caused in the TACI-Fc liquid candidate preparation (acetate, pH5.0, trehalose).This shows that trehalose has prevented the cohesion and the loss (far ultraviolet CD experimental results show that) of natural secondary structure effectively.

-exist in the preparation group of antiseptic, with regard to the cohesion rate, benzyl alcohol+benzalkonium chloride coupling provides optimum.

Conclusion

Several antibacterial have been estimated to TACI-Fc medicine (former batch dress) with the influence of protein integrity in the 100mg/ml TACI-Fc preparation of pH 5.0 sodium acetates and trehalose preparation.

Comprising any of these antibacterial all has negative effect to proteinic integrity, particularly to former batch powder charge product.0.3% benzyl alcohol+0.001% benzalkonium chloride coupling becomes protein structure illeffects minimum.

The stability of embodiment 3:TACI-Fc liquid candidate's preparation in the prefill syringe

Purpose

The purpose of this research is to be evaluated at 100mg/ml TACI-Fc liquid preparation (sodium acetate, trehalose, stability pH5.0) that fills in the 1ml Hypak syringe (W4023/50 and W4023/50G, FluroTec company) that clogs with two kinds of rubber closures.

Main result

TACI-Fc100mg/ml carries out 6 months detection in the 1ml glass syringe that clogs with coating stopper (W4023/50G Fluro Tec company) and no coating stopper (W4023/50G) to filling, and brief summary is as follows as a result:

The degradation rate of-dimer and HMW:40 ℃ (increasing by 1.6% weekly) and 25 ℃ (increasing by 0.5% in every month) is comparable.2-8 ℃ of observed performance is slightly different, though and the not obvious general stability that influences: two kinds of different product batch numbers increased by 0.2% and 0.1% in every month;

-protein content: protein content is not seen minimizing in storage period;

-pruning form: detect with bottle in the splicing form (comparing with medicine 2-8 ℃ the time does not increase, and 25 ℃ increase by 40% after 5 months approximately) of the comparable level of product;

-biopotency: biologic activity keeps up to 3 months (2-8 ℃ and 25 ℃);

-pH: do not see that pH changes storage period.

Conclusion

(sodium acetate+trehalose is stable pH5.0) to fill 100mg/ml TACI-Fc liquid preparation in 1ml Hypak syringe.Two kinds of rubber closures of this research evaluation (W4023/50 and W4023/50GFluroTec company) are of equal value, all do not influence the stability of this liquid preparation.

Embodiment 4: the stability that is contained in the varying strength ATACICEPT in the prefilled syringe

Assessed the stability of the varying strength atacicept that is contained in the prefilled syringe.Undertaken by method of reporting among the embodiment 1.

The compositions of given the test agent is as follows:

Mission Number	?Atacicept?mg/ml	Buffer	Trehalose dihydrate compound mg/ml
				??Atacicept?25/1	??25	The 10mM sodium acetate, pH 5	??88.4
??Atacicept?75/1	??75	The 10mM sodium acetate, pH 5	??88.4
				??Atacicept?150/1	??150	The 10mM sodium acetate, pH 5	??88.4
??Atacicept?25/1.2	??20.5	The 10mM sodium acetate, pH 5	??88.4
				??Atacicept?150/1.2	??125	The 10mM sodium acetate, pH 5	??88.4

Following table U-Z is seen in result's report of stability study.

The purity % that table U.SE-HPLC detects

Store 1,2,3,6,9,12 and after 18 months for 5 ℃:

Store 1,2,3 and after 6 months for 25 ℃:

The protein content (mg/ml) that Table V .SE-HPLC measures

Store 1,2,3,6,9,12 and after 18 months for 5 ℃:

	0 o'clock	1 month+5 ℃	2 months+5 ℃	3 months+5 ℃
					??Atacicept?25/1	??23.9	??27.9	??22.7	??21.9
??Atacicept?75/1	??75.0	??80.4	??77.9	??76.6
					??Atacicept?150/1	??150.0	??165.5	??160.2	??161.0
??Atacicept?25/1.2	??21.8	??24.3	??23.6	??22.0
					??Atacicept?150/1.2	??138.1	??138.4	??144.9	??148.5

	6 months+5 ℃	9 months+5 ℃	12 months+5 ℃	18 months+5 ℃
					??Atacicept?25/1	??25.1	??24.9	??26.1	??25.2
??Atacicept?75/1	??80.1	??78.8	??77.2
					??Atacicept?150/1	??156.5	??152.0	??157.9	??152.1
??Atacicept?25/1.2	??22.3	??21.3	??22.2	??21.6
					??Atacicept?150/1.2	??136.3	??134.1	??132.4	??126.1

Store 1,2,3 and after 6 months for 25 ℃

	0 o'clock	1 month+25 ℃	2 months+25 ℃	3 months+25 ℃	6 months+25 ℃
						??Atacicept?25/1	??23.9	??28.0	??21.5	??21.9	??25.1
??Atacicept?75/1	??75.0	??80.1	??77.4	??73.5	??79.8
						??Atacicept?150/1	??150.0	??163.8	??165.5	??162.8	??152.2
??Atacicept?25/1.2	??21.8	??22.9	??23.4	??21.3	??21.6
						??Atacicept?150/1.2	??138.1	??141.6	??137.2	??147.7	??128.6

After 40 ℃ of 1,2 and 4 weeks of storage

	0 o'clock	1 week+40 ℃	2 week+40 ℃	4 week+40 ℃
					??Atacicept?75/1	??75.0	??71.7	??71.9	??71.8

Table W. prunes form (%)

Store 1,2,3,6,9,12 and after 18 months for 5 ℃:

	0 o'clock	1 month+5 ℃	2 months+5 ℃	3 months+5 ℃
					??Atacicept?25/1	??11.0	??12.8	??12.8	??12.5
??Atacicept?75/1	??11.6	??13.8	??12.1	??13.0
					??Atacicept?150/1	??11.5	??12.3	??13.5	??13.1
??Atacicept?25/1.2	??12.7	??12.7	??12.8	??13.5
					??Atacicept?150/1.2	??12.3	??13.0	??12.5	??14.1

	6 months+5 ℃	9 months+5 ℃	12 months+5 ℃	18 months+5 ℃
					??Atacicept?25/1	??14.1	??14.7	??15.4	??18.4
??Atacicept?75/1	??13.5	??15.4	??16.7
					??Atacicept?150/1	??14.3	??16.1	??16.7	??19.3
??Atacicept?25/1.2	??15.9	??15.8	??16.9	??18.5
					??Atacicept?150/1.2	??15.4	??16.5	??18.6	??18.7

Store 1,2,3 and after 6 months for 25 ℃:

	0 o'clock	1 month+25 ℃	2 months+25 ℃	3 months+25 ℃	6 months+25 ℃
						??Atacicept?25/1	??11.0	??17.5	??20.1	??25.8	??32.8
??Atacicept?75/1	??11.6	??17.4	??20.4	??24.1	??33.8
						??Atacicept?150/1	??11.5	??18.3	??21.7	??26.7	??35.1
??Atacicept?25/1.2	??12.7	??16.7	??20.6	??25.4	??34.9

	0 o'clock	1 month+25 ℃	2 months+25 ℃	3 months+25 ℃	6 months+25 ℃
						??Atacicept?150/1.2	??12.3	??17.1	??22.1	??26.7	??37.3

The free Fc% that Table X .IEC-HPLC detects

Store 1,2,3,6,9,12 and after 18 months for 5 ℃:

	0 o'clock	1 month+5 ℃	2 months+5 ℃	3 months+5 ℃
					??Atacicept?25/1	??-	??0.08	??0.08	??0.08
??Atacicept?75/1	??0.09	??0.14	??0.14	??0.11
					??Atacicept?150/1	??-	??0.09	??0.09	??0.10
??Atacicept?25/1.2	??0.09	??0.08	??0.09	??0.09
					??Atacicept?150/1.2	??0.08	??0.09	??0.11	??0.13

	6 months+5 ℃	9 months+5 ℃	12 months+5 ℃	18 months+5 ℃
					??Atacicept?25/1	??0.18	??0.13	??0.12	??0.12
??Atacicept?75/1	??0.12	??0.13	??0.12
					??Atacicept?150/1	??0.19	??0.16	??0.15	??0.16
??Atacicept?25/1.2	??0.11	??0.11	??0.12	??0.11
					??Atacicept?150/1.2	??0.13	??0.17	??0.15	??0.16

Store 1,2,3 and after 6 months for 25 ℃:

	0 o'clock	1 month+25 ℃	2 months+25 ℃	3 months+25 ℃	6 months+25 ℃
						??Atacicept?25/1	??-	??0.17	??0.22	??0.40	??0.77
??Atacicept?75/1	??0.09	??0.25	??0.39	??0.43	??0.72
						??Atacicept?150/1	??-	??0.20	??0.32	??0.52	??0.87
??Atacicept?25/1.2	??0.09	??0.13	??0.30	??0.49	??0.70
						??Atacicept??150/1.2	??0.08	??0.18	??0.38	??0.51	??0.81

Table Y. biologic activity (U/ml)

Store 1,2,3,6,9,12 and after 18 months for 5 ℃:

	0 o'clock	1 month+5 ℃	2 months+5 ℃	3 months+5 ℃
					??Atacicept?25/1	??140213	??137160	??134504	??135542
??Atacicept?75/1	??423184	??410261	??379575	??374383
					??Atacicept?150/1	??836070	??774584	??754834	??819172
??Atacicept?25/1.2	??111981	??115990	??126041	??121648
					??Atacicept?150/1.2	??646679	??642858	??743090	??694864

	6 months+5 ℃	9 months+5 ℃	12 months+5 ℃	18 months+5 ℃
					??Atacicept?25/1	??126923	??147341	??130609	??108207
??Atacicept?75/1	??363668	??468484	??346080

	6 months+5 ℃	9 months+5 ℃	12 months+5 ℃	18 months+5 ℃
					??Atacicept?150/1	??814539	??843419	??809840	??565084
??Atacicept?25/1.2	??114946	??123750	??106004	??113312
					??Atacicept?150/1.2	??645404	??714223	??620301	??550851

Store 1,2,3 and after 6 months for 25 ℃:

	0 o'clock	1 month+25 ℃	2 months+25 ℃	3 months+25 ℃	6 months+25 ℃
						??Atacicept?25/1	??140213	??134212	??124601	??132349	??118224
??Atacicept?75/1	??423184	??387654	??336790	??365202	??327925
						??Atacicept?150/1	??836070	??776645	??719725	??760795	??677110
??Atacicept?25/1.2	??111981	??114068	??117615	??114296	??103328
						??Atacicept?150/1.2	??646679	??694993	??696945	??606957	??586586

Table Z.pH measures

Store 1,2 and after 3 months for 5 ℃:

	0 o'clock	1 month+5 ℃	2 months+5 ℃	3 months+5 ℃
					??Atacicept?25/1	??5.0	??5.0	??4.9	??4.9
??Atacicept?75/1	??5.1	??5.1	??5.1	??5.1
					??Atacicept?150/1	??5.0	??5.1	??5.0	??4.9
??Atacicept?25/1.2	??5.0	??4.9	??4.9	??4.8

	0 o'clock	1 month+5 ℃	2 months+5 ℃	3 months+5 ℃
					??Atacicept?150/1.2	??5.0	??5.0	??4.9	??4.9

	6 months+5 ℃	9 months+5 ℃	12 months+5 ℃	18 months+5 ℃
					??Atacicept?25/1	??5.0	??4.9	??5.1	??4.9
??Atacicept?75/1	??5.0	??5.1	??5.1
					??Atacicept?150/1	??5.1	??5.0	??5.2	??5.0
??Atacicept?25/1.2	??4.9	??4.9	??5.0	??5.0
					??Atacicept?150/1.2	??5.0	??5.0	??5.0	??5.0

Store 1,2,3 and after 6 months for 25 ℃:

	0 o'clock	1 month+25 ℃	2 months+25 ℃	3 months+25 ℃	6 months+25 ℃
						??Atacicept?25/1	??5.0	??4.9	??4.9	??4.9	??5.0
??Atacicept?75/1	??5.1	??5.1	??5.1	??5.1	??5.1
						??Atacicept?150/1	??5.0	??5.0	??5.0	??4.9	??5.1
??Atacicept?25/1.2	??5.0	??4.9	??4.9	??4.8	??4.9
						??Atacicept?150/1.2	??5.0	??5.0	??4.9	??4.9	??5.0

The preparation of embodiment 5.BLYS antagonist

The TACI-Fc that has prepared the terminal truncates of 4 seed amino acids.These the 4 kinds people tissue fibrinolysis zymogen activator sequences (disclosing) that all contain the modification of the amino acid residue 30 that is blended in SEQ IDNO:6 as WO 02/094852 (SEQ ID NO:25).Yet " Fc5 " in these 4 kinds of albumen is blended in the site difference of the TACI aminoacid sequence of SEQ ID NO:6.Table 1 has been summarized the structure of these 4 kinds of fusion rotein.

Table 1.TACI-Fc fusion rotein

The proteic title of TACI-Fc	The TACI amino acid residue
		??TACI(d1-29)-Fc5	The 30-154 aminoacid of SEQ ID NO:6
??TACI(d1-29，d107-154)-Fc5	The 30-106 aminoacid of SEQ ID NO:6
		??TACI(d1-29，d111-154)-Fc5	The 30-110 aminoacid of SEQ ID NO:6
??TACI(d1-29，d120-154)-Fc5	The 30-119 aminoacid of SEQ ID NO:6

Adopt standard technique by overlapping PCR prepared encoding proteins expression cassette (referring to, Horton etc. for example, 1989).The nucleic acid molecules that adopts the nucleic acid molecules of coding TACI and the Fc5 that encodes is as pcr template.Table 2 and 3 is the oligonucleotide primers of its evaluation.

Table 2. is used to prepare the oligonucleotide primers of TACI fusion rotein

The sequence of table 3. oligonucleotide

Primer	Nucleotide sequence	??SEQ?ID??NO.
			??ZC24，903	??5′TATTAGGCCGGCCACCATGGATGCAATGA?3′	??15
??ZC24，955	??5′TGAAGATTTGGGCTCCTTGAGACCTGGGA?3′	??16

Primer	Nucleotide sequence	??SEQ?ID??NO.
			??ZC24，952	??5′TCCCAGGTCTCAAGGAGCCCAAATCTTCA?3′	??17
??ZC24，946	??5′TAATTGGCGCGCCTCTAGATTATTTACCCGGAGACA?3′	??18
			??ZC24，951	??5′TGAAGATTTGGGCTCGTTCTCACAGAAGTA?3′	??19
??ZC24，949	??5′ATACTTCTGTGAGAACGAGCCCAAATCTTCA?3′	??20
			??ZC28，978	??5′TTTGGGCTCGCTCCTGAGCTTGTTCTCACA?3′	??21
??ZC28，979	??5′CTCAGGAGCGAGCCCAAATCTTCAGACA?3′	??22
			??ZC28，981	??5′TTTGGGCTCCCTGAGCTCTGGTGGAA?3′	??23
??ZC28，980	??5′GAGCTCAGGGAGCCCAAATCTTCAGACA?3′	??24

First round pcr amplification comprises 4 kinds of amino terminal truncate secondary response separately.Carry out this secondary response respectively, primary first-order equation is with 5 ' and 3 ' TACI oligonucleotide.Another secondary response 5 ' and 3 ' Fc5 oligonucleotide separately.The condition of first round pcr amplification is as follows: (2.5 units are Stratagene) to final volume 25 μ l to add the various 5 ' oligonucleotide of 20 μ M of 2.5mMdNTP, 0.5 μ l of about 200ng template DNA, 2.5 μ l 10xPfu reaction buffers (Stratagene), 2 μ l and 3 ' oligonucleotide and 0.5 μ l Pfu polymerase.The temperature of amplification be set to 94 ℃ 3 minutes, 35 take turns 94 ℃ 15 seconds, 50 ℃ 15 seconds, 72 ℃ 2 minutes, extended 2 minutes in 72 ℃ again.Use the agarose gel electrophoresis reaction product isolated, from gel, cut and reclaim according to manufacturers instruction corresponding to the band of estimating size with QIAGEN QIAQUICK gel extraction agent box (Qiagen company).

, carry out second and take turns PGR amplification or overlapping pcr amplification reaction as dna profiling with the fragment of first round PCR gel-purified.Second to take turns the condition of pcr amplification as follows: the 20 μ M ZC24 that add 2.5mMdNTP, the 0.5 μ l of separately template DNA of about 10ng TACI fragment and Fc5 fragment, 2.5 μ l 10xPfu reaction buffers (Stratagene), 2 μ l, 903 (SEQ ID NO:15) and ZC24, (2.5 units are Stratagene) to final volume 25 μ l for 946 (SEQ ID NO:18) and 0.5 μ l Pfu polymerase.The temperature of amplification be set to 94 ℃ 1 minute, 35 take turns 94 ℃ 15 seconds, 55 ℃ 15 seconds, 72 ℃ 2 minutes, 72 ℃ were extended 2 minutes again.Use the agarose gel electrophoresis reaction product isolated, from gel, cut and reclaim according to manufacturers instruction corresponding to the band of estimating size with QIAGENQIAQUICK gel extraction agent box (Qiagen company).

The TACI-Fc PCR product that separates 4 kinds of amino terminal truncates with the ZEROBLUNT TOPO PCR clone test kit of Invitrogen company by the scheme of manufacturer's recommendation respectively.Table 4 is nucleotide and aminoacid sequences of these TACI-Fc constructions.

The sequence of table 4.TACI-Fc variant

Behind the checking nucleotide sequence, digest the plasmid separately that comprises 4 kinds of amino terminal truncate TACI-Fc integrative nucleic acids, to discharge the section of coded amino acid with Fsel and Ascl.The Fsel-Ascl digestion fragment is connected in the mammalian expression vector that contains CMV promoter and the poly-A section of SV40.As described below with in this expression vector introducing Chinese hamster ovary cell.

Embodiment 6. usefulness Chinese hamster ovary cells are produced TACI-Fc albumen

Adapted to Chinese hamster ovary cell (CHO) the DG44 cell of suspension growth by electroporation with the transfection of TACI-Fc expression constructs, cultivated with the culture medium (Urlaub etc., 1986) of animal protein-free.CHO DG44 cell lacks functional dihydrofolate reductase gene owing to chromosomal dihydrofolate reductase has 2 topagnosises.Cultured cell in the presence of the progressive concentration methotrexate causes dihydrofolate reductase gene in this expression constructs and the amplification of the coding recombiant protein gene that is attached thereto.

The PFCHO culture medium (JRH Biosciences company, Lenexa, KS), in 4mM L-glutaminate (JRH Biosciences company) and the 1x hypoxanthine-thymidine additive (LifeTechnologies company) with CHO DG44 passage.Corning on the rotational oscillation platform shakes in the bottle in 37 ℃ and 5%CO ₂Rotate cultured cell with 120rpm under the condition.Use linearizing expression plasmid transfectional cell respectively.Aseptic for guaranteeing, the sub-carrier DNA of silicon milt (5 ' → 3 ' Inc.Boulder company that in Eppendorf tubule on ice, 200 μ g plasmid DNA and 20 μ L is sheared, 10mg/ml), 100% ethanol (the GoldShield Chemical company of the 3M NaOAc (pH5.2) of 22 μ L and 484 μ L, Hayward, CA) mix, carry out ethanol precipitation step 25 minute.After the cultivation, with being placed on 4 ℃ of microcentrifuges in the cold house in 14,000rpm is centrifugal with tubule, and abandoning supernatant uses 0.5ml 70% ethanol with washing of precipitate 2 times, air drying.

Precipitate the exsiccant while at DNA, with in the 25ml taper centrifuge tube altogether 10 ⁶Individual cell centrifugal 5 minutes in 900rpm, preparation CHO DG44 cell.CHO DG44 cell is resuspended in the PFCHO growth medium of cumulative volume 300 μ l, puts into the gene-pulse cuvette (Bio-Rad) of 0.4cm electrode gap.After the dry about 50 minutes clock times, DNA is resuspended to the PFCHO growth medium of 500 μ l, adds in the cell of cuvette, make cumulative volume be no more than 800 μ l, place following 5 minutes of room temperature to reduce bubble formation.Cuvette is placed BioRad gene pulse device II unit electroporation immediately, electroporation be set to 0.296kV (kilovolt) and 0.950HC (high capacitance).

The cell room temperature was cultivated 5 minutes, place the PFCHO culture medium of CoStar T-75 culture bottle cumulative volume 20ml then.Culture bottle is placed 37 ℃ and 5%CO ₂Cultivated 48 hours, with trypan blue exclusion dyeing with hemocytometer pair cell counting after, cell put into no hypoxanthine-thymidine additive and the PFCHO that contains 200mM methotrexate (Cal Biochem company) selects culture medium.

After methotrexate is selected to handle and reclaimed, contain the proteic conditioned medium of secreted TACI-Fc with Western printing and dyeing analytical review.

List of references

Altschul S F etc., J Mol Biol, 215,403-410,1990.

Altschul S F etc., NucleicAcids Res., 25:389-3402,1997.

Armour KL etc., 1999. " lack Fc γ receptor 1 combination and mononuclear cell and trigger active reorganization human IgG molecule " Eur J Immunol.29 (8): 2613-24.

Canfield SM, Morrison SL. " binding affinity of human IgG high affine Fc receptor with it is determined and be subjected to the adjusting of hinge region by a plurality of aminoacid in the CH2 district " .J Exp Med 1991; 173:1483-1491.

Cheema etc., Arthritis Rheum 2001; 44 (6): 1313-1319.

Devereux J etc., Nucleic Acids Res, 12,387-395,1984.

Do RKG, Hatada E, Lee H, Tourigny MR, Hilbert D, Chen-Kiang S. " based on the B cell stimulatory agents to the enhancing of humoral immunoresponse(HI) and apoptosis is weakened " .J Exp Med2000; 192 (7): 953-964.

Duncan etc., Nature 332:563 (1988).

Grantham etc., Science, Vol.185, pp.862-864 (1974).

Groom etc., J Clin Invest 2002; 109 (I): 59-68; Mariette X, Ann Rheum Dis2003; 62 (2): 168-171.

Gross etc., Nature 2000; 404:995-999.

Horton etc., Gene 77:61 (1989).

Kabat EA, Glusman M, Knaub V. " albumin and Y globulin with the immuno-chemical method detection by quantitative in the normal and pathology cerebrospinal fluid " .Am J Med 1948; 4:653-662.

Kabat, E.A., Wu, T.T., Perry, H.M., Gottesman, K.S., and Foeller, C. (1991), " sequence of immunology proteins of interest matter ". the 5th edition, NIH, Bethesda, MD.

Kalled SL. " effect of BAFF in immunologic function with autoimmune related " .Immunol Rev2005; 204:43-54.

Mackay etc., J Exp Me 1999; 190 (11); 1697-1710.

Marsters SA, Yan M, Pitti RM, Haas PE, Dixit VM, Ashkenazi A. " interaction of TNF congener BLyS and APRIL and TNF receptor homolog thing BCMA and TACI " .CurrBiol 2000; 10 (13): 785-788.

Moore etc., Science 1999; 285 (5425): 260-263; Schneider etc., J Exp Med1999; 189 (11): 1747-1756.

Do etc., J E * p Med 2000; 192 (7): 953-964.

Pearson，Methods?Enzymol.1990；183：63-98.

Roschke V, Sosnovtseva S, Ward CD, Hong JS, Smith R, Albert V etc., " expression of the heterogeneous trimer of biologic activity in general immune rheumatism disease patient that BLyS and APRIL form " .J Immunol 2002; 169:4314-4321.

Rudick etc., Neurology 2001; 56:1324-1330.

Schneider P, Mackay F, Steiner V, Hofmann K, Bodmer J-L, Holler N. " a kind of new part BAFF of tnf family cytokines can stimulate the growth of B cell " .J E * p Med1999; 189 (11): 1747-1756.

Shields RL. etc., 2001. " the high-resolution collection of illustrative plates of human IgG1's Fc γ RI, Fc γ RII, Fc γ RIII and FcRn binding site and have the design of the IgG1 variant of improved Fc γ R binding ability " .J Biol Chem.276 (9): 6591-604.

Soderstrom M, Link H, Xu Z, Fredriksson S. " optic nerve and multiple sclerosis: anti--MBP and the anti--accumulation of MBP peptide antibody secretory cell in CSF " .Neurology 1993; 43:1215-1222.

Sondermann etc., Nature 406:267 (2000).

Tao M-H, Smith RIF, Morrison S L. " architectural feature of human normal immunoglobulin IgG has determined the isotype difference of complement activation " .J Exp Med 1993; 178:661-667.

Thangarajh M, Gomes A, Masterman T, Hillert J, Hjelmstrom P. " B of TNF family cell activation factor (BAFF) and receptor expression thereof in the multiple sclerosis " JNeuroimmunol 2004; 152:183-190.

Thompson JS, Bixler SA, Qian F, Vora K, Scott ML, Cachero TG.BAFF-R, " a kind of new evaluation can with the interactional TNF receptor of BAFF specificity " Science 2001; 293:2108-2111.

Urlaub etc., Som.Cell.Molec.Genet.12:555 (1986).

Wines etc., J.Immunol.164:5313 (2000).

Sequence table

＜110〉Ares Trading S.A.

＜120〉TACI-immunoglobulin fusion protein formulations

<130>101966PWCN

 

<150>EP?07120489.5

<151>2007-11-12

 

<150>EP?07120490.3

<151>2007-11-12

 

<150>US?61/002,988

<151>2007-11-14

 

<150>US?61/003,028

<151>2007-11-14

 

<150>US?61/072,038

<151>2008-03-27

 

<150>EP?08005923.1

<151>2008-03-27

 

<160>25

<170>PatentIn?version?3.5

 

<210>1

<211>166

<212>PRT

＜213〉homo sapiens

 

<400>1

Met?Ser?Gly?Leu?Gly?Arg?Ser?Arg?Arg?Gly?Gly?Arg?Ser?Arg?Val?Asp

1???????????????5???????????????????10??????????????????15

Gln?Glu?Glu?Arg?Phe?Pro?Gln?Gly?Leu?Trp?Thr?Gly?Val?Ala?Met?Arg

20??????????????????25??????????????????30

Ser?Cys?Pro?Glu?Glu?Gln?Tyr?Trp?Asp?Pro?Leu?Leu?Gly?Thr?Cys?Met

35??????????????????40??????????????????45

Ser?Cys?Lys?Thr?Ile?Cys?Asn?His?Gln?Ser?Gln?Arg?Thr?Cys?Ala?Ala

50??????????????????55??????????????????60

Phe?Cys?Arg?Ser?Leu?Ser?Cys?Arg?Lys?Glu?Gln?Gly?Lys?Phe?Tyr?Asp

65??????????????????70??????????????????75??????????????????80

His?Leu?Leu?Arg?Asp?Cys?Ile?Ser?Cys?Ala?Ser?Ile?Cys?Gly?Gln?His

85??????????????????90??????????????????95

Pro?Lys?Gln?Cys?Ala?Tyr?Phe?Cys?Glu?Asn?Lys?Leu?Arg?Ser?Pro?Val

100?????????????????105?????????????????110

Asn?Leu?Pro?Pro?Glu?Leu?Arg?Arg?Gln?Arg?Ser?Gly?Glu?Val?Glu?Asn

115?????????????????120?????????????????125

Asn?Ser?Asp?Asn?Ser?Gly?Arg?Tyr?Gln?Gly?Leu?Gly?His?Arg?Gly?Ser

130?????????????????135?????????????????140

Glu?Ala?Ser?Pro?Ala?Leu?Pro?Gly?Leu?Lys?Leu?Ser?Ala?Asn?Gln?Val

145?????????????????150?????????????????155?????????????????160

Ala?Leu?Val?Tyr?Ser?Thr

165

 

<210>2

<211>232

<212>PRT

＜213〉homo sapiens

 

<400>2

Glu?Pro?Lys?Ser?Ser?Asp?Lys?Thr?His?Thr?Cys?Pro?Pro?Cys?Pro?Ala

1???????????????5???????????????????10??????????????????15

Pro?Glu?Ala?Glu?Gly?Ala?Pro?Ser?Val?Phe?Leu?Phe?Pro?Pro?Lys?Pro

20??????????????????25??????????????????30

Lys?Asp?Thr?Leu?Met?Ile?Ser?Arg?Thr?Pro?Glu?Val?Thr?Cys?Val?Val

35??????????????????40??????????????????45

Val?Asp?Val?Ser?His?Glu?Asp?Pro?Glu?Val?Lys?Phe?Asn?Trp?Tyr?Val

50??????????????????55??????????????????60

Asp?Gly?Val?Glu?Val?His?Asn?Ala?Lys?Thr?Lys?Pro?Arg?Glu?Glu?Gln

65??????????????????70??????????????????75??????????????????80

Tyr?Asn?Ser?Thr?Tyr?Arg?Val?Val?Ser?Val?Leu?Thr?Val?Leu?His?Gln

85??????????????????90??????????????????95

Asp?Trp?Leu?Asn?Gly?Lys?Glu?Tyr?Lys?Cys?Lys?Val?Ser?Asn?Lys?Ala

100?????????????????105?????????????????110

Leu?Pro?Ser?Ser?Ile?Glu?Lys?Thr?Ile?Ser?Lys?Ala?Lys?Gly?Gln?Pro

115?????????????????120?????????????????125

Arg?Glu?Pro?Gln?Val?Tyr?Thr?Leu?Pro?Pro?Ser?Arg?Asp?Glu?Leu?Thr

130?????????????????135?????????????????140

Lys?Asn?Gln?Val?Ser?Leu?Thr?Cys?Leu?Val?Lys?Gly?Phe?Tyr?Pro?Ser

145?????????????????150?????????????????155?????????????????160

Asp?Ile?Ala?Val?Glu?Trp?Glu?Ser?Asn?Gly?Gln?Pro?Glu?Asn?Asn?Tyr

165?????????????????170?????????????????175

Lys?Thr?Thr?Pro?Pro?Val?Leu?Asp?Ser?Asp?Gly?Ser?Phe?Phe?Leu?Tyr

180?????????????????185?????????????????190

Ser?Lys?Leu?Thr?Val?Asp?Lys?Ser?Arg?Trp?Gln?Gln?Gly?Asn?Val?Phe

195?????????????????200?????????????????205

Ser?Cys?Ser?Val?Met?His?Glu?Ala?Leu?His?Asn?His?Tyr?Thr?Gln?Lys

210?????????????????215?????????????????220

Ser?Leu?Ser?Leu?Ser?Pro?Gly?Lys

225?????????????????230

 

<210>3

<211>313

<212>PRT

＜213〉homo sapiens

 

<400>3

 

Ala?Met?Arg?Ser?Cys?Pro?Glu?Glu?Gln?Tyr?Trp?Asp?Pro?Leu?Leu?Gly

1???????????????5???????????????????10??????????????????15

Thr?Cys?Met?Ser?Cys?Lys?Thr?Ile?Cys?Asn?His?Gln?Ser?Gln?Arg?Thr

20??????????????????25??????????????????30

Cys?Ala?Ala?Phe?Cys?Arg?Ser?Leu?Ser?Cys?Arg?Lys?Glu?Gln?Gly?Lys

35??????????????????40??????????????????45

Phe?Tyr?Asp?His?Leu?Leu?Arg?Asp?Cys?Ile?Ser?Cys?Ala?Ser?Ile?Cys

50??????????????????55??????????????????60

Gly?Gln?His?Pro?Lys?Gln?Cys?Ala?Tyr?Phe?Cys?Glu?Asn?Lys?Leu?Arg

65??????????????????70??????????????????75??????????????????80

Ser?Glu?Pro?Lys?Ser?Ser?Asp?Lys?Thr?His?Thr?Cys?Pro?Pro?Cys?Pro

85??????????????????90??????????????????95

Ala?Pro?Glu?Ala?Glu?Gly?Ala?Pro?Ser?Val?Phe?Leu?Phe?Pro?Pro?Lys

100?????????????????105?????????????????110

Pro?Lys?Asp?Thr?Leu?Met?Ile?Ser?Arg?Thr?Pro?Glu?Val?Thr?Cys?Val

115?????????????????120?????????????????125

Val?Val?Asp?Val?Ser?His?Glu?Asp?Pro?Glu?Val?Lys?Phe?Asn?Trp?Tyr

130?????????????????135?????????????????140

Val?Asp?Gly?Val?Glu?Val?His?Asn?Ala?Lys?Thr?Lys?Pro?Arg?Glu?Glu

145?????????????????150?????????????????155?????????????????160

Gln?Tyr?Asn?Ser?Thr?Tyr?Arg?Val?Val?Ser?Val?Leu?Thr?Val?Leu?His

165?????????????????170?????????????????175

Gln?Asp?Trp?Leu?Asn?Gly?Lys?Glu?Tyr?Lys?Cys?Lys?Val?Ser?Asn?Lys

180?????????????????185?????????????????190

Ala?Leu?Pro?Ser?Ser?Ile?Glu?Lys?Thr?Ile?Ser?Lys?Ala?Lys?Gly?Gln

195?????????????????200?????????????????205

Pro?Arg?Glu?Pro?Gln?Val?Tyr?Thr?Leu?Pro?Pro?Ser?Arg?Asp?Glu?Leu

210?????????????????215?????????????????220

Thr?Lys?Asn?Gln?Val?Ser?Leu?Thr?Cys?Leu?Val?Lys?Gly?Phe?Tyr?Pro

225?????????????????230?????????????????235?????????????????240

Ser?Asp?Ile?Ala?Val?Glu?Trp?Glu?Ser?Asn?Gly?Gln?Pro?Glu?Asn?Asn

245?????????????????250?????????????????255

Tyr?Lys?Thr?Thr?Pro?Pro?Val?Leu?Asp?Ser?Asp?Gly?Ser?Phe?Phe?Leu

260?????????????????265?????????????????270

Tyr?Ser?Lys?Leu?Thr?Val?Asp?Lys?Ser?Arg?Trp?Gln?Gln?Gly?Asn?Val

275?????????????????280?????????????????285

Phe?Ser?Cys?Ser?Val?Met?His?Glu?Ala?Leu?His?Asn?His?Tyr?Thr?Gln

290?????????????????295?????????????????300

Lys?Ser?Leu?Ser?Leu?Ser?Pro?Gly?Lys

305?????????????????310

 

<210>4

<211>762

<212>DNA

＜213〉homo sapiens

 

<220>

<221>CDS

<222>(7)..(759)

<400>4

 

ggatcc?atg?aag?cac?ctg?tgg?ttc?ttc?ctc?ctg?ctg?gtg?gcg?gct?ccc??????48

Met?Lys?His?Leu?Trp?Phe?Phe?Leu?Leu?Leu?Val?Ala?Ala?Pro

1???????????????5???????????????????10

aga?tgg?gtc?ctg?tcc?gag?ccc?aaa?tct?tgt?gac?aaa?act?cac?aca?tgc?????96

Arg?Trp?Val?Leu?Ser?Glu?Pro?Lys?Ser?Cys?Asp?Lys?Thr?His?Thr?Cys

15??????????????????20??????????????????25??????????????????30

cca?ccg?tgc?cca?gca?cct?gaa?ctc?ctg?ggg?gga?ccg?tca?gtc?ttc?ctc????144

Pro?Pro?Cys?Pro?Ala?Pro?Glu?Leu?Leu?Gly?Gly?Pro?Ser?Val?Phe?Leu

35??????????????????40??????????????????45

ttc?ccc?cca?aaa?ccc?aag?gac?acc?ctc?atg?atc?tcc?cgg?acc?cct?gag????192

Phe?Pro?Pro?Lys?Pro?Lys?Asp?Thr?Leu?Met?Ile?Ser?Arg?Thr?Pro?Glu

50??????????????????55??????????????????60

gtc?aca?tgc?gtg?gtg?gtg?gac?gtg?agc?cac?gaa?gac?cct?gag?gtc?aag????240

Val?Thr?Cys?Val?Val?Val?Asp?Val?Ser?His?Glu?Asp?Pro?Glu?Val?Lys

65??????????????????70??????????????????75

ttc?aac?tgg?tac?gtg?gac?ggc?gtg?gag?gtg?cat?aat?gcc?aag?aca?aag????288

Phe?Asn?Trp?Tyr?Val?Asp?Gly?Val?Glu?Val?His?Asn?Ala?Lys?Thr?Lys

80??????????????????85??????????????????90

ccg?cgg?gag?gag?cag?tac?aac?agc?acg?tac?cgt?gtg?gtc?agc?gtc?ctc????336

Pro?Arg?Glu?Glu?Gln?Tyr?Asn?Ser?Thr?Tyr?Arg?Val?Val?Ser?Val?Leu

95??????????????????100?????????????????105?????????????????110

acc?gtc?ctg?cac?cag?gac?tgg?ctg?aat?ggc?aag?gag?tac?aag?tgc?aag????384

Thr?Val?Leu?His?Gln?Asp?Trp?Leu?Asn?Gly?Lys?Glu?Tyr?Lys?Cys?Lys

115?????????????????120?????????????????125

gtc?tcc?aac?aaa?gcc?ctc?cca?gcc?ccc?atc?gag?aaa?acc?atc?tcc?aaa????432

Val?Ser?Asn?Lys?Ala?Leu?Pro?Ala?Pro?Ile?Glu?Lys?Thr?Ile?Ser?Lys

130?????????????????135?????????????????140

gcc?aaa?ggg?cag?ccc?cga?gaa?cca?cag?gtg?tac?acc?ctg?ccc?cca?tcc????480

Ala?Lys?Gly?Gln?Pro?Arg?Glu?Pro?Gln?Val?Tyr?Thr?Leu?Pro?Pro?Ser

145?????????????????150?????????????????155

cgg?gat?gag?ctg?acc?aag?aac?cag?gtc?agc?ctg?acc?tgc?ctg?gtc?aaa????528

Arg?Asp?Glu?Leu?Thr?Lys?Asn?Gln?Val?Ser?Leu?Thr?Cys?Leu?Val?Lys

160?????????????????165?????????????????170

ggc?ttc?tat?ccc?agc?gac?atc?gcc?gtg?gag?tgg?gag?agc?aat?ggg?cag????576

Gly?Phe?Tyr?Pro?Ser?Asp?Ile?Ala?Val?Glu?Trp?Glu?Ser?Asn?Gly?Gln

175?????????????????180?????????????????185?????????????????190

ccg?gag?aac?aac?tac?aag?acc?acg?cct?ccc?gtg?ctg?gac?tcc?gac?ggc????624

Pro?Glu?Asn?Asn?Tyr?Lys?Thr?Thr?Pro?Pro?Val?Leu?Asp?Ser?Asp?Gly

195?????????????????200?????????????????205

tcc?ttc?ttc?ctc?tac?agc?aag?ctc?acc?gtg?gac?aag?agc?agg?tgg?cag????672

Ser?Phe?Phe?Leu?Tyr?Ser?Lys?Leu?Thr?Val?Asp?Lys?Ser?Arg?Trp?Gln

210?????????????????215?????????????????220

cag?ggg?aac?gtc?ttc?tca?tgc?tcc?gtg?atg?cat?gag?gct?ctg?cac?aac????720

Gln?Gly?Asn?Val?Phe?Ser?Cys?Ser?Val?Met?His?Glu?Ala?Leu?His?Asn

225?????????????????230?????????????????235

cac?tac?acg?cag?aag?agc?ctc?tcc?ctg?tct?ccg?ggt?aaa?tga????762

His?Tyr?Thr?Gln?Lys?Ser?Leu?Ser?Leu?Ser?Pro?Gly?Lys

240?????????????????245?????????????????250

 

<210>5

<211>251

<212>PRT

＜213〉homo sapiens

 

<400>5

 

Met?Lys?His?Leu?Trp?Phe?Phe?Leu?Leu?Leu?Val?Ala?Ala?Pro?Arg?Trp

1???????????????5???????????????????10??????????????????15

Val?Leu?Ser?Glu?Pro?Lys?Ser?Cys?Asp?Lys?Thr?His?Thr?Cys?Pro?Pro

20??????????????????25??????????????????30

Cys?Pro?Ala?Pro?Glu?Leu?Leu?Gly?Gly?Pro?Ser?Val?Phe?Leu?Phe?Pro

35??????????????????40??????????????????45

Pro?Lys?Pro?Lys?Asp?Thr?Leu?Met?Ile?Ser?Arg?Thr?Pro?Glu?Val?Thr

50??????????????????55?????????????????60

Cys?Val?Val?Val?Asp?Val?Ser?His?Glu?Asp?Pro?Glu?Val?Lys?Phe?Asn

65??????????????????70??????????????????75??????????????????80

Trp?Tyr?Val?Asp?Gly?Val?Glu?Val?His?Asn?Ala?Lys?Thr?Lys?Pro?Arg

85??????????????????90??????????????????95

Glu?Glu?Gln?Tyr?Asn?Ser?Thr?Tyr?Arg?Val?Val?Ser?Val?Leu?Thr?Val

100?????????????????105?????????????????110

Leu?His?Gln?Asp?Trp?Leu?Asn?Gly?Lys?Glu?Tyr?Lys?Cys?Lys?Val?Ser

115?????????????????120?????????????????125

Asn?Lys?Ala?Leu?Pro?Ala?Pro?Ile?Glu?Lys?Thr?Ile?Ser?Lys?Ala?Lys

130?????????????????135?????????????????140

Gly?Gln?Pro?Arg?Glu?Pro?Gln?Val?Tyr?Thr?Leu?Pro?Pro?Ser?Arg?Asp

145?????????????????150?????????????????155?????????????????160

Glu?Leu?Thr?Lys?Asn?Gln?Val?Ser?Leu?Thr?Cys?Leu?Val?Lys?Gly?Phe

165?????????????????170?????????????????175

Tyr?Pro?Ser?Asp?Ile?Ala?Val?Glu?Trp?Glu?Ser?Asn?Gly?Gln?Pro?Glu

180?????????????????185?????????????????190

Asn?Asn?Tyr?Lys?Thr?Thr?Pro?Pro?Val?Leu?Asp?Ser?Asp?Gly?Ser?Phe

195?????????????????200?????????????????205

Phe?Leu?Tyr?Ser?Lys?Leu?Thr?Val?Asp?Lys?Ser?Arg?Trp?Gln?Gln?Gly

210?????????????????215?????????????????220

Asn?Val?Phe?Ser?Cys?Ser?Val?Met?His?Glu?Ala?Leu?His?Asn?His?Tyr

225?????????????????230?????????????????235?????????????????240

Thr?Gln?Lys?Ser?Leu?Ser?Leu?Ser?Pro?Gly?Lys

245?????????????????250

 

<210>6

<211>293

<212>PRT

＜213〉homo sapiens

 

<400>6

 

Met?Ser?Gly?Leu?Gly?Arg?Ser?Arg?Arg?Gly?Gly?Arg?Ser?Arg?Val?Asp

1???????????????5???????????????????10??????????????????15

Gln?Glu?Glu?Arg?Phe?Pro?Gln?Gly?Leu?Trp?Thr?Gly?Val?Ala?Met?Arg

20??????????????????25??????????????????30

Ser?Cys?Pro?Glu?Glu?Gln?Tyr?Trp?Asp?Pro?Leu?Leu?Gly?Thr?Cys?Met

35??????????????????40??????????????????45

Ser?Cys?Lys?Thr?Ile?Cys?Asn?His?Gln?Ser?Gln?Arg?Thr?Cys?Ala?Ala

50??????????????????55??????????????????60

Phe?Cys?Arg?Ser?Leu?Ser?Cys?Arg?Lys?Glu?Gln?Gly?Lys?Phe?Tyr?Asp

65??????????????????70??????????????????75??????????????????80

His?Leu?Leu?Arg?Asp?Cys?Ile?Ser?Cys?Ala?Ser?Ile?Cys?Gly?Gln?His

85??????????????????90??????????????????95

Pro?Lys?Gln?Cys?Ala?Tyr?Phe?Cys?Glu?Asn?Lys?Leu?Arg?Ser?Pro?Val

100?????????????????105?????????????????110

Asn?Leu?Pro?Pro?Glu?Leu?Arg?Arg?Gln?Arg?Ser?Gly?Glu?Val?Glu?Asn

115?????????????????120?????????????????125

Asn?Ser?Asp?Asn?Ser?Gly?Arg?Tyr?Gln?Gly?Leu?Glu?His?Arg?Gly?Ser

130?????????????????135?????????????????140

Glu?Ala?Ser?Pro?Ala?Leu?Pro?Gly?Leu?Lys?Leu?Ser?Ala?Asp?Gln?Val

145?????????????????150?????????????????155?????????????????160

Ala?Leu?Val?Tyr?Ser?Thr?Leu?Gly?Leu?Cys?Leu?Cys?Ala?Val?Leu?Cys

165?????????????????170?????????????????175

Cys?Phe?Leu?Val?Ala?Val?Ala?Cys?Phe?Leu?Lys?Lys?Arg?Gly?Asp?Pro

180?????????????????185?????????????????190

Cys?Ser?Cys?Gln?Pro?Arg?Ser?Arg?Pro?Arg?Gln?Ser?Pro?Ala?Lys?Ser

195?????????????????200?????????????????205

Ser?Gln?Asp?His?Ala?Met?Glu?Ala?Gly?Ser?Pro?Val?Ser?Thr?Ser?Pro

210?????????????????215?????????????????220

Glu?Pro?Val?Glu?Thr?Cys?Ser?Phe?Cys?Phe?Pro?Glu?Cys?Arg?Ala?Pro

225?????????????????230?????????????????235?????????????????240

Thr?Gln?Glu?Ser?Ala?Val?Thr?Pro?Gly?Thr?Pro?Asp?Pro?Thr?Cys?Ala

245?????????????????250?????????????????255

Gly?Arg?Trp?Gly?Cys?His?Thr?Arg?Thr?Thr?Val?Leu?Gln?Pro?Cys?Pro

260?????????????????265?????????????????270

His?Ile?Pro?Asp?Ser?Gly?Leu?Gly?Ile?Val?Cys?Val?Pro?Ala?Gln?Glu

275?????????????????280?????????????????285

Gly?Gly?Pro?Gly?Ala

290

 

<210>7

<211>1214

<212>DNA

＜213〉homo sapiens

 

<220>

<221>CDS

<222>(17)..(1192)

 

<400>7

tattaggccg?gccacc?atg?gat?gca?atg?aag?aga?ggg?ctc?tgc?tgt?gtg?ctg????52

Met?Asp?Ala?Met?Lys?Arg?Gly?Leu?Cys?Cys?Val?Leu

1???????????????5???????????????????10

ctg?ctg?tgt?ggc?gcc?gtc?ttc?gtt?tcg?ctc?agc?cag?gaa?atc?cat?gcc????100

Leu?Leu?Cys?Gly?Ala?Val?Phe?Val?Ser?Leu?Ser?Gln?Glu?Ile?His?Ala

15??????????????????20??????????????????25

gag?ttg?aga?cgc?ttc?cgt?aga?gct?atg?aga?tcc?tgc?ccc?gaa?gag?cag????148

Glu?Leu?Arg?Arg?Phe?Arg?Arg?Ala?Met?Arg?Ser?Cys?Pro?Glu?Glu?Gln

30??????????????????35??????????????????40

tac?tgg?gat?cct?ctg?ctg?ggt?acc?tgc?atg?tcc?tgc?aaa?acc?att?tgc????196

Tyr?Trp?Asp?Pro?Leu?Leu?Gly?Thr?Cys?Met?Ser?Cys?Lys?Thr?Ile?Cys

45??????????????????50??????????????????55??????????????????60

aac?cat?cag?agc?cag?cgc?acc?tgt?gca?gcc?ttc?tgc?agg?tca?ctc?agc????244

Asn?His?Gln?Ser?Gln?Arg?Thr?Cys?Ala?Ala?Phe?Cys?Arg?Ser?Leu?Ser

65??????????????????70??????????????????75

tgc?cgc?aag?gag?caa?ggc?aag?ttc?tat?gac?cat?ctc?ctg?agg?gac?tgc????292

Cys?Arg?Lys?Glu?Gln?Gly?Lys?Phe?Tyr?Asp?His?Leu?Leu?Arg?Asp?Cys

80??????????????????85??????????????????90

atc?agc?tgt?gcc?tcc?atc?tgt?gga?cag?cac?cct?aag?caa?tgt?gca?tac????340

Ile?Ser?Cys?Ala?Ser?Ile?Cys?Gly?Gln?His?Pro?Lys?Gln?Cys?Ala?Tyr

95??????????????????100?????????????????105

ttc?tgt?gag?aac?aag?ctc?agg?agc?cca?gtg?aac?ctt?cca?cca?gag?ctc????388

Phe?Cys?Glu?Asn?Lys?Leu?Arg?Ser?Pro?Val?Asn?Leu?Pro?Pro?Glu?Leu

110?????????????????115?????????????????120

agg?aga?cag?cgg?agt?gga?gaa?gtt?gaa?aac?aat?tca?gac?aac?tcg?gga????436

Arg?Arg?Gln?Arg?Ser?Gly?Glu?Val?Glu?Asn?Asn?Ser?Asp?Asn?Ser?Gly

125?????????????????130?????????????????135?????????????????140

agg?tac?caa?gga?ttg?gag?cac?aga?ggc?tca?gaa?gca?agt?cca?gct?ctc????484

Arg?Tyr?Gln?Gly?Leu?Glu?His?Arg?Gly?Ser?Glu?Ala?Ser?Pro?Ala?Leu

145?????????????????150?????????????????155

cca?ggt?ctc?aag?gag?ccc?aaa?tct?tca?gac?aaa?act?cac?aca?tgc?cca????532

Pro?Gly?Leu?Lys?Glu?Pro?Lys?Ser?Ser?Asp?Lys?Thr?His?Thr?Cys?Pro

160?????????????????165?????????????????170

ccg?tgc?cca?gca?cct?gaa?gcc?gag?ggg?gca?ccg?tca?gtc?ttc?ctc?ttc????580

Pro?Cys?Pro?Ala?Pro?Glu?Ala?Glu?Gly?Ala?Pro?Ser?Val?Phe?Leu?Phe

175?????????????????180?????????????????185

ccc?cca?aaa?ccc?aag?gac?acc?ctc?atg?atc?tcc?cgg?acc?cct?gag?gtc????628

Pro?Pro?Lys?Pro?Lys?Asp?Thr?Leu?Met?Ile?Ser?Arg?Thr?Pro?Glu?Val

190?????????????????195?????????????????200

aca?tgc?gtg?gtg?gtg?gac?gtg?agc?cac?gaa?gac?cct?gag?gtc?aag?ttc????676

Thr?Cys?Val?Val?Val?Asp?Val?Ser?His?Glu?Asp?Pro?Glu?Val?Lys?Phe

205?????????????????210?????????????????215?????????????????220

aac?tgg?tac?gtg?gac?ggc?gtg?gag?gtg?cat?aat?gcc?aag?aca?aag?ccg????724

Asn?Trp?Tyr?Val?Asp?Gly?Val?Glu?Val?His?Asn?Ala?Lys?Thr?Lys?Pro

225?????????????????230?????????????????235

cgg?gag?gag?cag?tac?aac?agc?acg?tac?cgt?gtg?gtc?agc?gtc?ctc?acc???????772

Arg?Glu?Glu?Gln?Tyr?Asn?Ser?Thr?Tyr?Arg?Val?Val?Ser?Val?Leu?Thr

240?????????????????245?????????????????250

gtc?ctg?cac?cag?gac?tgg?ctg?aat?ggc?aag?gag?tac?aag?tgc?aag?gtc???????820

Val?Leu?His?Gln?Asp?Trp?Leu?Asn?Gly?Lys?Glu?Tyr?Lys?Cys?Lys?Val

255?????????????????260?????????????????265

tcc?aac?aaa?gcc?ctc?cca?tcc?tcc?atc?gag?aaa?acc?atc?tcc?aaa?gcc???????868

Ser?Asn?Lys?Ala?Leu?Pro?Ser?Ser?Ile?Glu?Lys?Thr?Ile?Ser?Lys?Ala

270?????????????????275?????????????????280

aaa?ggg?cag?ccc?cga?gaa?cca?cag?gtg?tac?acc?ctg?ccc?cca?tcc?cgg???????916

Lys?Gly?Gln?Pro?Arg?Glu?Pro?Gln?Val?Tyr?Thr?Leu?Pro?Pro?Ser?Arg

285?????????????????290?????????????????295?????????????????300

gat?gag?ctg?acc?aag?aac?cag?gtc?agc?ctg?acc?tgc?ctg?gtc?aaa?ggc???????964

Asp?Glu?Leu?Thr?Lys?Asn?Gln?Val?Ser?Leu?Thr?Cys?Leu?Val?Lys?Gly

305?????????????????310?????????????????315

ttc?tat?ccc?agc?gac?atc?gcc?gtg?gag?tgg?gag?agc?aat?ggg?cag?ccg??????1012

Phe?Tyr?Pro?Ser?Asp?Ile?Ala?Val?Glu?Trp?Glu?Ser?Asn?Gly?Gln?Pro

320?????????????????325?????????????????330

gag?aac?aac?tac?aag?acc?acg?cct?ccc?gtg?ctg?gac?tcc?gac?ggc?tcc??????1060

Glu?Asn?Asn?Tyr?Lys?Thr?Thr?Pro?Pro?Val?Leu?Asp?Ser?Asp?Gly?Ser

335?????????????????340?????????????????345

ttc?ttc?ctc?tac?agc?aag?ctc?acc?gtg?gac?aag?agc?agg?tgg?cag?cag??????1108

Phe?Phe?Leu?Tyr?Ser?Lys?Leu?Thr?Val?Asp?Lys?Ser?Arg?Trp?Gln?Gln

350?????????????????355?????????????????360

ggg?aac?gtc?ttc?tca?tgc?tcc?gtg?atg?cat?gag?gct?ctg?cac?aac?cac??????1156

Gly?Asn?Val?Phe?Ser?Cys?Ser?Val?Met?His?Glu?Ala?Leu?His?Asn?His

365?????????????????370?????????????????375?????????????????380

tac?acg?cag?aag?agc?ctc?tcc?ctg?tct?ccg?ggt?aaa?taatctagag???????????1202

Tyr?Thr?Gln?Lys?Ser?Leu?Ser?Leu?Ser?Pro?Gly?Lys

385?????????????????390

gcgcgccaat?ta????????????????????????????????????????????????????????1214

 

<210>8

<211>392

<212>PRT

＜213〉homo sapiens

 

<400>8

 

Met?Asp?Ala?Met?Lys?Arg?Gly?Leu?Cys?Cys?Val?Leu?Leu?Leu?Cys?Gly

1???????????????5???????????????????10??????????????????15

Ala?Val?Phe?Val?Ser?Leu?Ser?Gln?Glu?Ile?His?Ala?Glu?Leu?Arg?Arg

20??????????????????25??????????????????30

Phe?Arg?Arg?Ala?Met?Arg?Ser?Cys?Pro?Glu?Glu?Gln?Tyr?Trp?Asp?Pro

35??????????????????40??????????????????45

Leu?Leu?Gly?Thr?Cys?Met?Ser?Cys?Lys?Thr?Ile?Cys?Asn?His?Gln?Ser

50??????????????????55??????????????????60

Gln?Arg?Thr?Cys?Ala?Ala?Phe?Cys?Arg?Ser?Leu?Ser?Cys?Arg?Lys?Glu

65??????????????????70??????????????????75??????????????????80

Gln?Gly?Lys?Phe?Tyr?Asp?His?Leu?Leu?Arg?Asp?Cys?Ile?Ser?Cys?Ala

85??????????????????90??????????????????95

Ser?Ile?Cys?Gly?Gln?His?Pro?Lys?Gln?Cys?Ala?Tyr?Phe?Cys?Glu?Asn

100?????????????????105?????????????????110

Lys?Leu?Arg?Ser?Pro?Val?Asn?Leu?Pro?Pro?Glu?Leu?Arg?Arg?Gln?Arg

115?????????????????120?????????????????125

Ser?Gly?Glu?Val?Glu?Asn?Asn?Ser?Asp?Asn?Ser?Gly?Arg?Tyr?Gln?Gly

130?????????????????135?????????????????140

Leu?Glu?His?Arg?Gly?Ser?Glu?Ala?Ser?Pro?Ala?Leu?Pro?Gly?Leu?Lys

145?????????????????150?????????????????155?????????????????160

Glu?Pro?Lys?Ser?Ser?Asp?Lys?Thr?His?Thr?Cys?Pro?Pro?Cys?Pro?Ala

165?????????????????170?????????????????175

Pro?Glu?Ala?Glu?Gly?Ala?Pro?Ser?Val?Phe?Leu?Phe?Pro?Pro?Lys?Pro

180?????????????????185?????????????????190

Lys?Asp?Thr?Leu?Met?Ile?Ser?Arg?Thr?Pro?Glu?Val?Thr?Cys?Val?Val

195?????????????????200?????????????????205

Val?Asp?Val?Ser?His?Glu?Asp?Pro?Glu?Val?Lys?Phe?Asn?Trp?Tyr?Val

210?????????????????215?????????????????220

Asp?Gly?Val?Glu?Val?His?Asn?Ala?Lys?Thr?Lys?Pro?Arg?Glu?Glu?Gln

225?????????????????230?????????????????235?????????????????240

Tyr?Asn?Ser?Thr?Tyr?Arg?Val?Val?Ser?Val?Leu?Thr?Val?Leu?His?Gln

245?????????????????250?????????????????255

Asp?Trp?Leu?Asn?Gly?Lys?Glu?Tyr?Lys?Cys?Lys?Val?Ser?Asn?Lys?Ala

260?????????????????265?????????????????270

Leu?Pro?Ser?Ser?Ile?Glu?Lys?Thr?Ile?Ser?Lys?Ala?Lys?Gly?Gln?Pro

275?????????????????280?????????????????285

Arg?Glu?Pro?Gln?Val?Tyr?Thr?Leu?Pro?Pro?Ser?Arg?Asp?Glu?Leu?Thr

290?????????????????295?????????????????300

Lys?Asn?Gln?Val?Ser?Leu?Thr?Cys?Leu?Val?Lys?Gly?Phe?Tyr?Pro?Ser

305?????????????????310?????????????????315?????????????????320

Asp?Ile?Ala?Val?Glu?Trp?Glu?Ser?Asn?Gly?Gln?Pro?Glu?Asn?Asn?Tyr

325?????????????????330?????????????????335

Lys?Thr?Thr?Pro?Pro?Val?Leu?Asp?Ser?Asp?Gly?Ser?Phe?Phe?Leu?Tyr

340?????????????????345?????????????????350

Ser?Lys?Leu?Thr?Val?Asp?Lys?Ser?Arg?Trp?Gln?Gln?Gly?Asn?Val?Phe

355?????????????????360?????????????????365

Ser?Cys?Ser?Val?Met?His?Glu?Ala?Leu?His?Asn?His?Tyr?Thr?Gln?Lys

370?????????????????375?????????????????380

Ser?Leu?Ser?Leu?Ser?Pro?Gly?Lys

385?????????????????390

 

<210>9

<211>1070

<212>DNA

＜213〉homo sapiens

 

<220>

<221>CDS

<222>(17)..(1048)

 

<400>9

 

tattaggccg?gccacc?atg?gat?gca?atg?aag?aga?ggg?ctc?tgc?tgt?gtg?ctg???52

Met?Asp?Ala?Met?Lys?Arg?Gly?Leu?Cys?Cys?Val?Leu

1???????????????5???????????????????10

ctg?ctg?tgt?ggc?gcc?gtc?ttc?gtt?tcg?ctc?agc?cag?gaa?atc?cat?gcc????100

Leu?Leu?Cys?Gly?Ala?Val?Phe?Val?Ser?Leu?Ser?Gln?Glu?Ile?His?Ala

15??????????????????20??????????????????25

gag?ttg?aga?cgc?ttc?cgt?aga?gct?atg?aga?tcc?tgc?ccc?gaa?gag?cag????148

Glu?Leu?Arg?Arg?Phe?Arg?Arg?Ala?Met?Arg?Ser?Cys?Pro?Glu?Glu?Gln

30??????????????????35??????????????????40

tac?tgg?gat?cct?ctg?ctg?ggt?acc?tgc?atg?tcc?tgc?aaa?acc?att?tgc????196

Tyr?Trp?Asp?Pro?Leu?Leu?Gly?Thr?Cys?Met?Ser?Cys?Lys?Thr?Ile?Cys

45??????????????????50??????????????????55??????????????????60

aac?cat?cag?agc?cag?cgc?acc?tgt?gca?gcc?ttc?tgc?agg?tca?ctc?agc????244

Asn?His?Gln?Ser?Gln?Arg?Thr?Cys?Ala?Ala?Phe?Cys?Arg?Ser?Leu?Ser

65??????????????????70??????????????????75

tgc?cgc?aag?gag?caa?ggc?aag?ttc?tat?gac?cat?ctc?ctg?agg?gac?tgc????292

Cys?Arg?Lys?Glu?Gln?Gly?Lys?Phe?Tyr?Asp?His?Leu?Leu?Arg?Asp?Cys

80??????????????????85??????????????????90

atc?agc?tgt?gcc?tcc?atc?tgt?gga?cag?cac?cct?aag?caa?tgt?gca?tac????340

Ile?Ser?Cys?Ala?Ser?Ile?Cys?Gly?Gln?His?Pro?Lys?Gln?Cys?Ala?Tyr

95??????????????????100?????????????????105

ttc?tgt?gag?aac?gag?ccc?aaa?tct?tca?gac?aaa?act?cac?aca?tgc?cca????388

Phe?Cys?Glu?Asn?Glu?Pro?Lys?Ser?Ser?Asp?Lys?Thr?His?Thr?Cys?Pro

110?????????????????115?????????????????120

ccg?tgc?cca?gca?cct?gaa?gcc?gag?ggg?gca?ccg?tca?gtc?ttc?ctc?ttc????436

Pro?Cys?Pro?Ala?Pro?Glu?Ala?Glu?Gly?Ala?Pro?Ser?Val?Phe?Leu?Phe

125?????????????????130?????????????????135?????????????????140

ccc?cca?aaa?ccc?aag?gac?acc?ctc?atg?atc?tcc?cgg?acc?cct?gag?gtc????484

Pro?Pro?Lys?Pro?Lys?Asp?Thr?Leu?Met?Ile?Ser?Arg?Thr?Pro?Glu?Val

145?????????????????150?????????????????155

aca?tgc?gtg?gtg?gtg?gac?gtg?agc?cac?gaa?gac?cct?gag?gtc?aag?ttc???????532

Thr?Cys?Val?Val?Val?Asp?Val?Ser?His?Glu?Asp?Pro?Glu?Val?Lys?Phe

160?????????????????165?????????????????170

aac?tgg?tac?gtg?gac?ggc?gtg?gag?gtg?cat?aat?gcc?aag?aca?aag?ccg???????580

Asn?Trp?Tyr?Val?Asp?Gly?Val?Glu?Val?His?Asn?Ala?Lys?Thr?Lys?Pro

175?????????????????180?????????????????185

cgg?gag?gag?cag?tac?aac?agc?acg?tac?cgt?gtg?gtc?agc?gtc?ctc?acc???????628

Arg?Glu?Glu?Gln?Tyr?Asn?Ser?Thr?Tyr?Arg?Val?Val?Ser?Val?Leu?Thr

190?????????????????195?????????????????200

gtc?ctg?cac?cag?gac?tgg?ctg?aat?ggc?aag?gag?tac?aag?tgc?aag?gtc???????676

Val?Leu?His?Gln?Asp?Trp?Leu?Asn?Gly?Lys?Glu?Tyr?Lys?Cys?Lys?Val

205?????????????????210?????????????????215?????????????????220

tcc?aac?aaa?gcc?ctc?cca?tcc?tcc?atc?gag?aaa?acc?atc?tcc?aaa?gcc???????724

Ser?Asn?Lys?Ala?Leu?Pro?Ser?Ser?Ile?Glu?Lys?Thr?Ile?Ser?Lys?Ala

225?????????????????230?????????????????235

aaa?ggg?cag?ccc?cga?gaa?cca?cag?gtg?tac?acc?ctg?ccc?cca?tcc?cgg???????772

Lys?Gly?Gln?Pro?Arg?Glu?Pro?Gln?Val?Tyr?Thr?Leu?Pro?Pro?Ser?Arg

240?????????????????245?????????????????250

gat?gag?ctg?acc?aag?aac?cag?gtc?agc?ctg?acc?tgc?ctg?gtc?aaa?ggc???????820

Asp?Glu?Leu?Thr?Lys?Asn?Gln?Val?Ser?Leu?Thr?Cys?Leu?Val?Lys?Gly

255?????????????????260?????????????????265

ttc?tat?ccc?agc?gac?atc?gcc?gtg?gag?tgg?gag?agc?aat?ggg?cag?ccg???????868

Phe?Tyr?Pro?Ser?Asp?Ile?Ala?Val?Glu?Trp?Glu?Ser?Asn?Gly?Gln?Pro

270?????????????????275?????????????????280

gag?aac?aac?tac?aag?acc?acg?cct?ccc?gtg?ctg?gac?tcc?gac?ggc?tcc???????916

Glu?Asn?Asn?Tyr?Lys?Thr?Thr?Pro?Pro?Val?Leu?Asp?Ser?Asp?Gly?Ser

285?????????????????290?????????????????295?????????????????300

ttc?ttc?ctc?tac?agc?aag?ctc?acc?gtg?gac?aag?agc?agg?tgg?cag?cag???????964

Phe?Phe?Leu?Tyr?Ser?Lys?Leu?Thr?Val?Asp?Lys?Ser?Arg?Trp?Gln?Gln

305?????????????????310?????????????????315

ggg?aac?gtc?ttc?tca?tgc?tcc?gtg?atg?cat?gag?gct?ctg?cac?aac?cac??????1012

Gly?Asn?Val?Phe?Ser?Cys?Ser?Val?Met?His?Glu?Ala?Leu?His?Asn?His

320?????????????????325?????????????????330

tac?acg?cag?aag?agc?ctc?tcc?ctg?tct?ccg?ggt?aaa?taatctagag???????????1058

Tyr?Thr?Gln?Lys?Ser?Leu?Ser?Leu?Ser?Pro?Gly?Lys

335?????????????????340

gcgcgccaat?ta????????????????????????????????????????????????????????1070

 

<210>10

<211>344

<212>PRT

＜213〉homo sapiens

 

<400>10

 

Met?Asp?Ala?Met?Lys?Arg?Gly?Leu?Cys?Cys?Val?Leu?Leu?Leu?Cys?Gly

1???????????????5???????????????????10??????????????????15

Ala?Val?Phe?Val?Ser?Leu?Ser?Gln?Glu?Ile?His?Ala?Glu?Leu?Arg?Arg

20??????????????????25??????????????????30

Phe?Arg?Arg?Ala?Met?Arg?Ser?Cys?Pro?Glu?Glu?Gln?Tyr?Trp?Asp?Pro

35??????????????????40??????????????????45

Leu?Leu?Gly?Thr?Cys?Met?Ser?Cys?Lys?Thr?Ile?Cys?Asn?His?Gln?Ser

50??????????????????55??????????????????60

Gln?Arg?Thr?Cys?Ala?Ala?Phe?Cys?Arg?Ser?Leu?Ser?Cys?Arg?Lys?Glu

65??????????????????70??????????????????75??????????????????80

Gln?Gly?Lys?Phe?Tyr?Asp?His?Leu?Leu?Arg?Asp?Cys?Ile?Ser?Cys?Ala

85??????????????????90??????????????????95

Ser?Ile?Cys?Gly?Gln?His?Pro?Lys?Gln?Cys?Ala?Tyr?Phe?Cys?Glu?Asn

100?????????????????105?????????????????110

Glu?Pro?Lys?Ser?Ser?Asp?Lys?Thr?His?Thr?Cys?Pro?Pro?Cys?Pro?Ala

115?????????????????120?????????????????125

Pro?Glu?Ala?Glu?Gly?Ala?Pro?Ser?Val?Phe?Leu?Phe?Pro?Pro?Lys?Pro

130?????????????????135?????????????????140

Lys?Asp?Thr?Leu?Met?Ile?Ser?Arg?Thr?Pro?Glu?Val?Thr?Cys?Val?Val

145?????????????????150?????????????????155?????????????????160

Val?Asp?Val?Ser?His?Glu?Asp?Pro?Glu?Val?Lys?Phe?Asn?Trp?Tyr?Val

165?????????????????170?????????????????175

Asp?Gly?Val?Glu?Val?His?Asn?Ala?Lys?Thr?Lys?Pro?Arg?Glu?Glu?Gln

180?????????????????185?????????????????190

Tyr?Asn?Ser?Thr?Tyr?Arg?Val?Val?Ser?Val?Leu?Thr?Val?Leu?His?Gln

195?????????????????200?????????????????205

Asp?Trp?Leu?Asn?Gly?Lys?Glu?Tyr?Lys?Cys?Lys?Val?Ser?Asn?Lys?Ala

210?????????????????215?????????????????220

Leu?Pro?Ser?Ser?Ile?Glu?Lys?Thr?Ile?Ser?Lys?Ala?Lys?Gly?Gln?Pro

225?????????????????230?????????????????235?????????????????240

Arg?Glu?Pro?Gln?Val?Tyr?Thr?Leu?Pro?Pro?Ser?Arg?Asp?Glu?Leu?Thr

245?????????????????250?????????????????255

Lys?Asn?Gln?Val?Ser?Leu?Thr?Cys?Leu?Val?Lys?Gly?Phe?Tyr?Pro?Ser

260?????????????????265?????????????????270

Asp?Ile?Ala?Val?Glu?Trp?Glu?Ser?Asn?Gly?Gln?Pro?Glu?Asn?Asn?Tyr

275?????????????????280?????????????????285

Lys?Thr?Thr?Pro?Pro?Val?Leu?Asp?Ser?Asp?Gly?Ser?Phe?Phe?Leu?Tyr

290?????????????????295?????????????????300

Ser?Lys?Leu?Thr?Val?Asp?Lys?Ser?Arg?Trp?Gln?Gln?Gly?Asn?Val?Phe

305?????????????????310?????????????????315?????????????????320

Ser?Cys?Ser?Val?Met?His?Glu?Ala?Leu?His?Asn?His?Tyr?Thr?Gln?Lys

325?????????????????330?????????????????335

Ser?Leu?Ser?Leu?Ser?Pro?Gly?Lys

340

 

<210>11

<211>1082

<212>DNA

＜213〉homo sapiens

 

<220>

<221>CDS

<222>(17)..(1060)

 

<400>11

tattaggccg?gccacc?atg?gat?gca?atg?aag?aga?ggg?ctc?tgc?tgt?gtg?ctg???52

Met?Asp?Ala?Met?Lys?Arg?Gly?Leu?Cys?Cys?Val?Leu

1???????????????5???????????????????10

ctg?ctg?tgt?ggc?gcc?gtc?ttc?gtt?tcg?ctc?agc?cag?gaa?atc?cat?gcc????100

Leu?Leu?Cys?Gly?Ala?Val?Phe?Val?Ser?Leu?Ser?Gln?Glu?Ile?His?Ala

15??????????????????20??????????????????25

gag?ttg?aga?cgc?ttc?cgt?aga?gct?atg?aga?tcc?tgc?ccc?gaa?gag?cag????148

Glu?Leu?Arg?Arg?Phe?Arg?Arg?Ala?Met?Arg?Ser?Cys?Pro?Glu?Glu?Gln

30??????????????????35??????????????????40

tac?tgg?gat?cct?ctg?ctg?ggt?acc?tgc?atg?tcc?tgc?aaa?acc?att?tgc????196

Tyr?Trp?Asp?Pro?Leu?Leu?Gly?Thr?Cys?Met?Ser?Cys?Lys?Thr?Ile?Cys

45??????????????????50??????????????????55??????????????????60

aac?cat?cag?agc?cag?cgc?acc?tgt?gca?gcc?ttc?tgc?agg?tca?ctc?agc????244

Asn?His?Gln?Ser?Gln?Arg?Thr?Cys?Ala?Ala?Phe?Cys?Arg?Ser?Leu?Ser

65??????????????????70??????????????????75

tgc?cgc?aag?gag?caa?ggc?aag?ttc?tat?gac?cat?ctc?ctg?agg?gac?tgc????292

Cys?Arg?Lys?Glu?Gln?Gly?Lys?Phe?Tyr?Asp?His?Leu?Leu?Arg?Asp?Cys

80??????????????????85??????????????????90

atc?agc?tgt?gcc?tcc?atc?tgt?gga?cag?cac?cct?aag?caa?tgt?gca?tac????340

Ile?Ser?Cys?Ala?Ser?Ile?Cys?Gly?Gln?His?Pro?Lys?Gln?Cys?Ala?Tyr

95??????????????????100?????????????????105

ttc?tgt?gag?aac?aag?ctc?agg?agc?gag?ccc?aaa?tct?tca?gac?aaa?act????388

Phe?Cys?Glu?Asn?Lys?Leu?Arg?Ser?Glu?Pro?Lys?Ser?Ser?Asp?Lys?Thr

110?????????????????115?????????????????120

cac?aca?tgc?cca?ccg?tgc?cca?gca?cct?gaa?gcc?gag?ggg?gca?ccg?tca????436

His?Thr?Cys?Pro?Pro?Cys?Pro?Ala?Pro?Glu?Ala?Glu?Gly?Ala?Pro?Ser

125?????????????????130?????????????????135?????????????????140

gtc?ttc?ctc?ttc?ccc?cca?aaa?ccc?aag?gac?acc?ctc?atg?atc?tcc?cgg????484

Val?Phe?Leu?Phe?Pro?Pro?Lys?Pro?Lys?Asp?Thr?Leu?Met?Ile?Ser?Arg

145?????????????????150?????????????????155

acc?cct?gag?gtc?aca?tgc?gtg?gtg?gtg?gac?gtg?agc?cac?gaa?gac?cct?????532

Thr?Pro?Glu?Val?Thr?Cys?Val?Val?Val?Asp?Val?Ser?His?Glu?Asp?Pro

160?????????????????165?????????????????170

gag?gtc?aag?ttc?aac?tgg?tac?gtg?gac?ggc?gtg?gag?gtg?cat?aat?gcc?????580

Glu?Val?Lys?Phe?Asn?Trp?Tyr?Val?Asp?Gly?Val?Glu?Val?His?Asn?Ala

175?????????????????180?????????????????185

aag?aca?aag?ccg?cgg?gag?gag?cag?tac?aac?agc?acg?tac?cgt?gtg?gtc?????628

Lys?Thr?Lys?Pro?Arg?Glu?Glu?Gln?Tyr?Asn?Ser?Thr?Tyr?Arg?Val?Val

190?????????????????195?????????????????200

agc?gtc?ctc?acc?gtc?ctg?cac?cag?gac?tgg?ctg?aat?ggc?aag?gag?tac?????676

Ser?Val?Leu?Thr?Val?Leu?His?Gln?Asp?Trp?Leu?Asn?Gly?Lys?Glu?Tyr

205?????????????????210?????????????????215?????????????????220

aag?tgc?aag?gtc?tcc?aac?aaa?gcc?ctc?cca?tcc?tcc?atc?gag?aaa?acc?????724

Lys?Cys?Lys?Val?Ser?Asn?Lys?Ala?Leu?Pro?Ser?Ser?Ile?Glu?Lys?Thr

225?????????????????230?????????????????235

atc?tcc?aaa?gcc?aaa?ggg?cag?ccc?cga?gaa?cca?cag?gtg?tac?acc?ctg?????772

Ile?Ser?Lys?Ala?Lys?Gly?Gln?Pro?Arg?Glu?Pro?Gln?Val?Tyr?Thr?Leu

240?????????????????245?????????????????250

ccc?cca?tcc?cgg?gat?gag?ctg?acc?aag?aac?cag?gtc?agc?ctg?acc?tgc?????820

Pro?Pro?Ser?Arg?Asp?Glu?Leu?Thr?Lys?Asn?Gln?Val?Ser?Leu?Thr?Cys

255?????????????????260?????????????????265

ctg?gtc?aaa?ggc?ttc?tat?ccc?agc?gac?atc?gcc?gtg?gag?tgg?gag?agc?????868

Leu?Val?Lys?Gly?Phe?Tyr?Pro?Ser?Asp?Ile?Ala?Val?Glu?Trp?Glu?Ser

270?????????????????275?????????????????280

aat?ggg?cag?ccg?gag?aac?aac?tac?aag?acc?acg?cct?ccc?gtg?ctg?gac?????916

Asn?Gly?Gln?Pro?Glu?Asn?Asn?Tyr?Lys?Thr?Thr?Pro?Pro?Val?Leu?Asp

285?????????????????290?????????????????295?????????????????300

tcc?gac?ggc?tcc?ttc?ttc?ctc?tac?agc?aag?ctc?acc?gtg?gac?aag?agc?????964

Ser?Asp?Gly?Ser?Phe?Phe?Leu?Tyr?Ser?Lys?Leu?Thr?Val?Asp?Lys?Ser

305?????????????????310?????????????????315

agg?tgg?cag?cag?ggg?aac?gtc?ttc?tca?tgc?tcc?gtg?atg?cat?gag?gct????1012

Arg?Trp?Gln?Gln?Gly?Asn?Val?Phe?Ser?Cys?Ser?Val?Met?His?Glu?Ala

320?????????????????325?????????????????330

ctg?cac?aac?cac?tac?acg?cag?aag?agc?ctc?tcc?ctg?tct?ccg?ggt?aaa????1060

Leu?His?Asn?His?Tyr?Thr?Gln?Lys?Ser?Leu?Ser?Leu?Ser?Pro?Gly?Lys

335?????????????????340?????????????????345

taatctagag?gcgcgccaat?ta???????????????????????????????????????????1082

 

<210>12

<211>348

<212>PRT

＜213〉homo sapiens

 

<400>12

 

Met?Asp?Ala?Met?Lys?Arg?Gly?Leu?Cys?Cys?Val?Leu?Leu?Leu?Cys?Gly

1???????????????5???????????????????10???????????????????15

Ala?Val?Phe?Val?Ser?Leu?Ser?Gln?Glu?Ile?His?Ala?Glu?Leu?Arg?Arg

20??????????????????25??????????????????30

Phe?Arg?Arg?Ala?Met?Arg?Ser?Cys?Pro?Glu?Glu?Gln?Tyr?Trp?Asp?Pro

35??????????????????40??????????????????45

Leu?Leu?Gly?Thr?Cys?Met?Ser?Cys?Lys?Thr?Ile?Cys?Asn?His?Gln?Ser

50??????????????????55??????????????????60

Gln?Arg?Thr?Cys?Ala?Ala?Phe?Cys?Arg?Ser?Leu?Ser?Cys?Arg?Lys?Glu

65??????????????????70??????????????????75??????????????????80

Gln?Gly?Lys?Phe?Tyr?Asp?His?Leu?Leu?Arg?Asp?Cys?Ile?Ser?Cys?Ala

85??????????????????90??????????????????95

Ser?Ile?Cys?Gly?Gln?His?Pro?Lys?Gln?Cys?Ala?Tyr?Phe?Cys?Glu?Asn

100?????????????????105?????????????????110

Lys?Leu?Arg?Ser?Glu?Pro?Lys?Ser?Ser?Asp?Lys?Thr?His?Thr?Cys?Pro

115?????????????????120?????????????????125

Pro?Cys?Pro?Ala?Pro?Glu?Ala?Glu?Gly?Ala?Pro?Ser?Val?Phe?Leu?Phe

130?????????????????135?????????????????140

Pro?Pro?Lys?Pro?Lys?Asp?Thr?Leu?Met?Ile?Ser?Arg?Thr?Pro?Glu?Val

145?????????????????150?????????????????155?????????????????160

Thr?Cys?Val?Val?Val?Asp?Val?Ser?His?Glu?Asp?Pro?Glu?Val?Lys?Phe

165?????????????????170?????????????????175

Asn?Trp?Tyr?Val?Asp?Gly?Val?Glu?Val?His?Asn?Ala?Lys?Thr?Lys?Pro

180?????????????????185?????????????????190

Arg?Glu?Glu?Gln?Tyr?Asn?Ser?Thr?Tyr?Arg?Val?Val?Ser?Val?Leu?Thr

195?????????????????200?????????????????205

Val?Leu?His?Gln?Asp?Trp?Leu?Asn?Gly?Lys?Glu?Tyr?Lys?Cys?Lys?Val

210?????????????????215?????????????????220

Ser?Asn?Lys?Ala?Leu?Pro?Ser?Ser?Ile?Glu?Lys?Thr?Ile?Ser?Lys?Ala

225?????????????????230?????????????????235?????????????????240

Lys?Gly?Gln?Pro?Arg?Glu?Pro?Gln?Val?Tyr?Thr?Leu?Pro?Pro?Ser?Arg

245?????????????????250?????????????????255

Asp?Glu?Leu?Thr?Lys?Asn?Gln?Val?Ser?Leu?Thr?Cys?Leu?Val?Lys?Gly

260?????????????????265?????????????????270

Phe?Tyr?Pro?Ser?Asp?Ile?Ala?Val?Glu?Trp?Glu?Ser?Asn?Gly?Gln?Pro

275?????????????????280?????????????????285

Glu?Asn?Asn?Tyr?Lys?Thr?Thr?Pro?Pro?Val?Leu?Asp?Ser?Asp?Gly?Ser

290?????????????????295?????????????????300

Phe?Phe?Leu?Tyr?Ser?Lys?Leu?Thr?Val?Asp?Lys?Ser?Arg?Trp?Gln?Gln

305?????????????????310?????????????????315?????????????????320

Gly?Asn?Val?Phe?Ser?Cys?Ser?Val?Met?His?Glu?Ala?Leu?His?Asn?His

325?????????????????330?????????????????335

Tyr?Thr?Gln?Lys?Ser?Leu?Ser?Leu?Ser?Pro?Gly?Lys

340?????????????????345

 

<210>13

<211>1109

<212>DNA

＜213〉homo sapiens

 

<220>

<221>CDS

<222>(17)..(1090)

 

<400>13

 

tattaggccg?gccacc?atg?gat?gca?atg?aag?aga?ggg?ctc?tgc?tgt?gtg?ctg???52

Met?Asp?Ala?Met?Lys?Arg?Gly?Leu?Cys?Cys?Val?Leu

1???????????????5???????????????????10

ctg?ctg?tgt?ggc?gcc?gtc?ttc?gtt?tcg?ctc?agc?cag?gaa?atc?cat?gcc????100

Leu?Leu?Cys?Gly?Ala?Val?Phe?Val?Ser?Leu?Ser?Gln?Glu?Ile?His?Ala

15??????????????????20??????????????????25

gag?ttg?aga?cgc?ttc?cgt?aga?gct?atg?aga?tcc?tgc?ccc?gaa?gag?cag????148

Glu?Leu?Arg?Arg?Phe?Arg?Arg?Ala?Met?Arg?Ser?Cys?Pro?Glu?Glu?Gln

30??????????????????35??????????????????40

tac?tgg?gat?cct?ctg?ctg?ggt?acc?tgc?atg?tcc?tgc?aaa?acc?att?tgc????196

Tyr?Trp?Asp?Pro?Leu?Leu?Gly?Thr?Cys?Met?Ser?Cys?Lys?Thr?Ile?Cys

45??????????????????50??????????????????55??????????????????60

aac?cat?cag?agc?cag?cgc?acc?tgt?gca?gcc?ttc?tgc?agg?tca?ctc?agc????244

Asn?His?Gln?Ser?Gln?Arg?Thr?Cys?Ala?Ala?Phe?Cys?Arg?Ser?Leu?Ser

65??????????????????70??????????????????75

tgc?cgc?aag?gag?caa?ggc?aag?ttc?tat?gac?cat?ctc?ctg?agg?gac?tgc????292

Cys?Arg?Lys?Glu?Gln?Gly?Lys?Phe?Tyr?Asp?His?Leu?Leu?Arg?Asp?Cys

80??????????????????85??????????????????90

atc?agc?tgt?gcc?tcc?atc?tgt?gga?cag?cac?cct?aag?caa?tgt?gca?tac????340

Ile?Ser?Cys?Ala?Ser?Ile?Cys?Gly?Gln?His?Pro?Lys?Gln?Cys?Ala?Tyr

95??????????????????100?????????????????105

ttc?tgt?gag?aac?aag?ctc?agg?agc?cca?gtg?aac?ctt?cca?cca?gag?ctc????388

Phe?Cys?Glu?Asn?Lys?Leu?Arg?Ser?Pro?Val?Asn?Leu?Pro?Pro?Glu?Leu

110?????????????????115?????????????????120

agg?gag?ccc?aaa?tct?tca?gac?aaa?act?cac?aca?tgc?cca?ccg?tgc?cca????436

Arg?Glu?Pro?Lys?Ser?Ser?Asp?Lys?Thr?His?Thr?Cys?Pro?Pro?Cys?Pro

125?????????????????130?????????????????135?????????????????140

gca?cct?gaa?gcc?gag?ggg?gca?ccg?tca?gtc?ttc?ctc?ttc?ccc?cca?aaa?????484

Ala?Pro?Glu?Ala?Glu?Gly?Ala?Pro?Ser?Val?Phe?Leu?Phe?Pro?Pro?Lys

145?????????????????150?????????????????155

ccc?aag?gac?acc?ctc?atg?atc?tcc?cgg?acc?cct?gag?gtc?aca?tgc?gtg?????532

Pro?Lys?Asp?Thr?Leu?Met?Ile?Ser?Arg?Thr?Pro?Glu?Val?Thr?Cys?Val

160?????????????????165?????????????????170

gtg?gtg?gac?gtg?agc?cac?gaa?gac?cct?gag?gtc?aag?ttc?aac?tgg?tac?????580

Val?Val?Asp?Val?Ser?His?Glu?Asp?Pro?Glu?Val?Lys?Phe?Asn?Trp?Tyr

175?????????????????180?????????????????185

gtg?gac?ggc?gtg?gag?gtg?cat?aat?gcc?aag?aca?aag?ccg?cgg?gag?gag?????628

Val?Asp?Gly?Val?Glu?Val?His?Asn?Ala?Lys?Thr?Lys?Pro?Arg?Glu?Glu

190?????????????????195?????????????????200

cag?tac?aac?agc?acg?tac?cgt?gtg?gtc?agc?gtc?ctc?acc?gtc?ctg?cac?????676

Gln?Tyr?Asn?Ser?Thr?Tyr?Arg?Val?Val?Ser?Val?Leu?Thr?Val?Leu?His

205?????????????????210?????????????????215?????????????????220

cag?gac?tgg?ctg?aat?ggc?aag?gag?tac?aag?tgc?aag?gtc?tcc?aac?aaa?????724

Gln?Asp?Trp?Leu?Asn?Gly?Lys?Glu?Tyr?Lys?Cys?Lys?Val?Ser?Asn?Lys

225?????????????????230?????????????????235

gcc?ctc?cca?tcc?tcc?atc?gag?aaa?acc?atc?tcc?aaa?gcc?aaa?ggg?cag?????772

Ala?Leu?Pro?Ser?Ser?Ile?Glu?Lys?Thr?Ile?Ser?Lys?Ala?Lys?Gly?Gln

240?????????????????245?????????????????250

ccc?cga?gaa?cca?cag?gtg?tac?acc?ctg?ccc?cca?tcc?cgg?gat?gag?ctg?????820

Pro?Arg?Glu?Pro?Gln?Val?Tyr?Thr?Leu?Pro?Pro?Ser?Arg?Asp?Glu?Leu

255?????????????????260?????????????????265

acc?aag?aac?cag?gtc?agc?ctg?acc?tgc?ctg?gtc?aaa?ggc?ttc?tat?ccc?????868

Thr?Lys?Asn?Gln?Val?Ser?Leu?Thr?Cys?Leu?Val?Lys?Gly?Phe?Tyr?Pro

270?????????????????275?????????????????280

agc?gac?atc?gcc?gtg?gag?tgg?gag?agc?aat?ggg?cag?ccg?gag?aac?aac?????916

Ser?Asp?Ile?Ala?Val?Glu?Trp?Glu?Ser?Asn?Gly?Gln?Pro?Glu?Asn?Asn

285?????????????????290?????????????????295?????????????????300

tac?aag?acc?acg?cct?ccc?gtg?ctg?gac?tcc?gac?ggc?tcc?ttc?ttc?ctc?????964

Tyr?Lys?Thr?Thr?Pro?Pro?Val?Leu?Asp?Ser?Asp?Gly?Ser?Phe?Phe?Leu

305?????????????????310?????????????????315

tac?agc?aag?ctc?acc?gtg?gac?aag?agc?agg?tgg?cag?cag?ggg?aac?gtc????1012

Tyr?Ser?Lys?Leu?Thr?Val?Asp?Lys?Ser?Arg?Trp?Gln?Gln?Gly?Asn?Val

320?????????????????325?????????????????330

ttc?tca?tgc?tcc?gtg?atg?cat?gag?gct?ctg?cac?aac?cac?tac?acg?cag????1060

Phe?Ser?Cys?Ser?Val?Met?His?Glu?Ala?Leu?His?Asn?His?Tyr?Thr?Gln

335?????????????????340?????????????????345

aag?agc?ctc?tcc?ctg?tct?ccg?ggt?aaa?taa?tctagaggcg?cgccaatta???????1109

Lys?Ser?Leu?Ser?Leu?Ser?Pro?Gly?Lys

350?????????????????355

<210>14

<211>357

<212>PRT

＜213〉homo sapiens

 

<400>14

 

Met?Asp?Ala?Met?Lys?Arg?Gly?Leu?Cys?Cys?Val?Leu?Leu?Leu?Cys?Gly

1???????????????5???????????????????10??????????????????15

Ala?Val?Phe?Val?Ser?Leu?Ser?Gln?Glu?Ile?His?Ala?Glu?Leu?Arg?Arg

20??????????????????25??????????????????30

Phe?Arg?Arg?Ala?Met?Arg?Ser?Cys?Pro?Glu?Glu?Gln?Tyr?Trp?Asp?Pro

35??????????????????40??????????????????45

Leu?Leu?Gly?Thr?Cys?Met?Ser?Cys?Lys?Thr?Ile?Cys?Asn?His?Gln?Ser

50??????????????????55??????????????????60

Gln?Arg?Thr?Cys?Ala?Ala?Phe?Cys?Arg?Ser?Leu?Ser?Cys?Arg?Lys?Glu

65??????????????????70??????????????????75??????????????????80

Gln?Gly?Lys?Phe?Tyr?Asp?His?Leu?Leu?Arg?Asp?Cys?Ile?Ser?Cys?Ala

85??????????????????90??????????????????95

Ser?Ile?Cys?Gly?Gln?His?Pro?Lys?Gln?Cys?Ala?Tyr?Phe?Cys?Glu?Asn

100?????????????????105?????????????????110

Lys?Leu?Arg?Ser?Pro?Val?Asn?Leu?Pro?Pro?Glu?Leu?Arg?Glu?Pro?Lys

115?????????????????120?????????????????125

Ser?Ser?Asp?Lys?Thr?His?Thr?Cys?Pro?Pro?Cys?Pro?Ala?Pro?Glu?Ala

130?????????????????135?????????????????140

Glu?Gly?Ala?Pro?Ser?Val?Phe?Leu?Phe?Pro?Pro?Lys?Pro?Lys?Asp?Thr

145?????????????????150?????????????????155?????????????????160

Leu?Met?Ile?Ser?Arg?Thr?Pro?Glu?Val?Thr?Cys?Val?Val?Val?Asp?Val

165?????????????????170?????????????????175

Ser?His?Glu?Asp?Pro?Glu?Val?Lys?Phe?Asn?Trp?Tyr?Val?Asp?Gly?Val

180?????????????????185?????????????????190

Glu?Val?His?Asn?Ala?Lys?Thr?Lys?Pro?Arg?Glu?Glu?Gln?Tyr?Asn?Ser

195?????????????????200?????????????????205

Thr?Tyr?Arg?Val?Val?Ser?Val?Leu?Thr?Val?Leu?His?Gln?Asp?Trp?Leu

210?????????????????215?????????????????220

Asn?Gly?Lys?Glu?Tyr?Lys?Cys?Lys?Val?Ser?Asn?Lys?Ala?Leu?Pro?Ser

225?????????????????230?????????????????235?????????????????240

Ser?Ile?Glu?Lys?Thr?Ile?Ser?Lys?Ala?Lys?Gly?Gln?Pro?Arg?Glu?Pro

245?????????????????250?????????????????255

Gln?Val?Tyr?Thr?Leu?Pro?Pro?Ser?Arg?Asp?Glu?Leu?Thr?Lys?Asn?Gln

260?????????????????265?????????????????270

Val?Ser?Leu?Thr?Cys?Leu?Val?Lys?Gly?Phe?Tyr?Pro?Ser?Asp?Ile?Ala

275?????????????????280?????????????????285

Val?Glu?Trp?Glu?Ser?Asn?Gly?Gln?Pro?Glu?Asn?Asn?Tyr?Lys?Thr?Thr

290?????????????????295?????????????????300

Pro?Pro?Val?Leu?Asp?Ser?Asp?Gly?Ser?Phe?Phe?Leu?Tyr?Ser?Lys?Leu

305?????????????????310?????????????????315?????????????????320

Thr?Val?Asp?Lys?Ser?Arg?Trp?Gln?Gln?Gly?Asn?Val?Phe?Ser?Cys?Ser

325?????????????????330?????????????????335

Val?Met?His?Glu?Ala?Leu?His?Asn?His?Tyr?Thr?Gln?Lys?Ser?Leu?Ser

340?????????????????345?????????????????350

Leu?Ser?Pro?Gly?Lys

355

 

<210>15

<211>29

<212>DNA

＜213〉artificial sequence

 

<220>

＜223〉primer sequence

 

<400>15

tattaggccg?gccaccatgg?atgcaatga??????????????????????????????29

 

<210>16

<211>29

<212>DNA

＜213〉artificial sequence

 

<220>

＜223〉primer sequence

 

<400>16

tgaagatttg?ggctccttga?gacctggga??????????????????????????????29

 

<210>17

<211>29

<212>DNA

＜213〉artificial sequence

 

<220>

＜223〉primer sequence

 

<400>17

tcccaggtct?caaggagccc?aaatcttca?????????????????????29

 

<210>18

<211>36

<212>DNA

＜213〉artificial sequence

 

<220>

＜223〉primer sequence

 

<400>18

taattggcgc?gcctctagat?tatttacccg?gagaca?????????????36

 

<210>19

<211>30

<212>DNA

＜213〉artificial sequence

 

<220>

＜223〉primer sequence

 

<400>19

tgaagatttg?ggctcgttct?cacagaagta????????????????????30

 

<210>20

<211>31

<212>DNA

＜213〉artificial sequence

 

<220>

＜223〉primer sequence

 

<400>20

atacttctgt?gagaacgagc?ccaaatcttc?a??????????????????31

 

<210>21

<211>30

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer sequence

 

<400>21

tttgggctcg?ctcctgagct?tgttctcaca????????????30

 

<210>22

<211>28

<212>DNA

＜213〉artificial sequence

 

<220>

＜223〉primer sequence

 

<400>22

ctcaggagcg?agcccaaatc?ttcagaca??????????????28

 

<210>23

<211>26

<212>DNA

＜213〉artificial sequence

 

<220>

＜223〉primer sequence

 

<400>23

tttgggctcc?ctgagctctg?gtggaa????????????????26

 

<210>24

<211>28

<212>DNA

＜213〉artificial sequence

 

<220>

＜223〉primer sequence

 

<400>24

gagctcaggg?agcccaaatc?ttcagaca??????????????28

 

<210>25

<211>35

<212>PRT

＜213〉artificial sequence

 

<220>

＜223〉signal peptide sequence of Xiu Shiing

<400>25

 

Met?Asp?Ala?Met?Lys?Arg?Gly?Leu?Cys?Cys?Val?Leu?Leu?Leu?Cys?Gly

1???????????????5???????????????????10??????????????????15

Ala?Val?Phe?Val?Ser?Leu?Ser?Gln?Glu?Ile?His?Ala?Glu?Leu?Arg?Arg

20??????????????????25??????????????????30

Phe?Arg?Arg

35

Claims (23)

1. preparation comprises:

A) TACI-immunoglobulin (TACI-Ig) fusion rotein, it comprises:

The ectodomain of i.TACI or its can be in conjunction with fragment or the variants of BLyS and/or APRIL; With

Ii. the constant region of immunoglobulin; With

B) make the pH scope of preparation maintain buffer between the 4.5-5.5.

2. preparation as claimed in claim 1, its pH scope is 4.7-5.3, or 4.9-5.1, or pH5.0.

3. preparation as claimed in claim 1 or 2, wherein said buffer is selected from: acetate, phosphate, succinate, citrate and histidine buffering liquid.

4. as any one described preparation in the above claim, wherein said acetate buffer is a sodium acetate.

5. preparation as claimed in claim 4, the concentration of wherein said sodium acetate is 5-25mM.

6. as any one described preparation in the above claim, said preparation also comprises trehalose.

7. preparation as claimed in claim 6, the concentration range of wherein said trehalose are 60-100mg/ml.

8. preparation as claimed in claim 7, the concentration of wherein said TACI-Ig fusion rotein are between 70-100mg/ml, and the concentration of described trehalose is between 80-100mg/m, and described buffer is the 10mM sodium acetate buffer.

9. as any one described preparation in the above claim, said preparation also comprises antiseptic.

10. preparation as claimed in claim 9, wherein said antiseptic are benzyl alcohol associating benzalkonium chlorides.

11. as any one described preparation in the above claim, wherein said TACI-Ig fragment comprises amino acid residue 34-66 and/or the amino acid residue 71-104 of SEQ ID NO:1.

12. preparation as claimed in claim 11, wherein said fragment comprise the amino acid residue 30-110 of SEQ ID NO:1, or at least 90% identical or be less than the variant of 10 conservative amino acid substitutions with it, described variant can be in conjunction with BlyS and/or APRIL.

13. as any one described preparation in the above claim, wherein said constant region for immunoglobulin is from human IgG1's constant region.

14. preparation as claimed in claim 13, wherein said human IgG1's constant region comprises SEQ IDNO:2 sequence, or it contains the variant that is less than 20 conservative amino acid substitutions.

15. as the described preparation of above any one claim, said preparation comprises SEQ ID NO:3 sequence, or at least 90% identical or be less than the variant of 30 conservative amino acid substitutions with it, described variant can be in conjunction with BlyS and/or APRIL.

16. as the described preparation of above any one claim, the described TACI-Ig fusion rotein concentration range that said preparation comprises is 20-180mg/ml.

17. as the described preparation of above any one claim, wherein said preparation is a liquid dosage form.

18. as the described preparation of above any one claim, wherein said preparation is the multiple dose administration preparation.

19. pharmaceutical composition that comprises above any one described preparation of claim.

20. as any one described preparation of claim 1-18 or the described pharmaceutical composition of claim 19, described preparation or pharmaceutical composition are used for the treatment of autoimmune disease or lymphocytic hyperplasia disease.

21. a method for preparing any one described preparation of claim 1-18, this method comprise preparation TACI-Ig fusion rotein liquid solution and regulate the pH to 4.5-5.5 of described liquid solution or the step of 4.7-5.3 or 4.9-5.1 scope or pH5.0.

22. the method for preparing preparation as claimed in claim 21, described method comprise that the described preparation with scheduled volume is placed on the step in the sterilization container.

23. method as claimed in claim 22, wherein said container is selected from the syringe of vial or prefill.